U.S. patent application number 16/489876 was filed with the patent office on 2020-01-09 for pyridyl derivatives as bromodomain inhibitors.
The applicant listed for this patent is GLAXOSMITHKLINE INTELLECTUAL PROPERTY (NO.2) LIMITED. Invention is credited to Stephen John ATKINSON, Emmanuel Hubert DEMONT, Lee Andrew HARRISON, Etienne LEVERNIER, Alexander G. PRESTON, Jonathan Thomas SEAL, Ian David WALL, Robert J. WATSON, James Michael WOOLVEN.
Application Number | 20200009140 16/489876 |
Document ID | / |
Family ID | 58544313 |
Filed Date | 2020-01-09 |
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United States Patent
Application |
20200009140 |
Kind Code |
A1 |
ATKINSON; Stephen John ; et
al. |
January 9, 2020 |
PYRIDYL DERIVATIVES AS BROMODOMAIN INHIBITORS
Abstract
The present invention is directed to pyridyl derivatives which
are bromodomain inhibitors, pharmaceutical compositions comprising
the compounds and the use of the compounds or the compositions in
the treatment of various diseases or conditions
Inventors: |
ATKINSON; Stephen John;
(Stevenage, Hertfordshire, GB) ; DEMONT; Emmanuel
Hubert; (Stevenage, Hertfordshire, GB) ; HARRISON;
Lee Andrew; (Stevenage, Hertfordshire, GB) ;
LEVERNIER; Etienne; (Stevenage, Hertfordshire, GB) ;
PRESTON; Alexander G.; (Stevenage, Hertfordshire, GB)
; SEAL; Jonathan Thomas; (Stevenage, Hertfordshire,
GB) ; WALL; Ian David; (Stevenage, Hertfordshire,
GB) ; WATSON; Robert J.; (Stevenage, Hertfordshire,
GB) ; WOOLVEN; James Michael; (Stevenage,
Hertfordshire, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE INTELLECTUAL PROPERTY (NO.2) LIMITED |
Brentford, Middlesex |
|
GB |
|
|
Family ID: |
58544313 |
Appl. No.: |
16/489876 |
Filed: |
February 27, 2018 |
PCT Filed: |
February 27, 2018 |
PCT NO: |
PCT/EP2018/054730 |
371 Date: |
August 29, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 401/06 20130101;
A61K 31/501 20130101; C07D 413/12 20130101; C07D 401/12 20130101;
A61P 37/00 20180101; A61K 31/44 20130101; A61K 31/444 20130101;
C07D 213/81 20130101; A61K 31/4439 20130101; C07D 401/14 20130101;
A61K 31/506 20130101; C07D 417/12 20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 31/44 20060101 A61K031/44; C07D 213/81 20060101
C07D213/81; A61K 31/4439 20060101 A61K031/4439; C07D 401/12
20060101 C07D401/12; C07D 413/12 20060101 C07D413/12; A61K 31/444
20060101 A61K031/444; A61K 31/501 20060101 A61K031/501; C07D 417/12
20060101 C07D417/12; C07D 401/14 20060101 C07D401/14 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 2017 |
GB |
1703282.2 |
Claims
1.-30. (canceled)
31. A compound of Formula (I) ##STR00103## or a pharmaceutically
acceptable salt thereof wherein: R.sup.1 is --C.sub.1-3alkyl or
cyclopropyl; R.sup.2 is H, --CH.sub.3, C.sub.2-6alkyl optionally
substituted by one, two, three, four, or five fluoro,
--C.sub.2-6alkylOR.sup.7, --C.sub.2-6alkylNR.sup.7R.sup.8,
--(CH.sub.2).sub.mSO.sub.2C.sub.1-3alkyl,
--(CH.sub.2).sub.mC(O)NR.sup.7R.sup.8, --(CH.sub.2).sub.mCN,
--(CH.sub.2).sub.mCO.sub.2R.sup.7,
--(CH.sub.2).sub.mNHCO.sub.2C(CH.sub.3).sub.3, or R.sup.2 is
--(CH.sub.2).sub.nC.sub.5-6heteroaryl wherein C.sub.5-6heteroaryl
is optionally substituted by one or two substituents independently
selected from halo, --C.sub.1-4alkyl, --C.sub.3-4cycloalkyl, and
--C.sub.0-4alkylOR.sup.5; R.sup.3 is H, --C.sub.1-4alkyl,
cyclopropyl, fluoro, chloro, --CH.sub.2F, --C.sub.0-3alkylOR.sup.5,
or --C.sub.0-3alkylCN; R.sup.4 is phenyl or a heteroaryl group
wherein each are optionally substituted by one, two, or three
R.sup.6 groups which may be the same or different; R.sup.5 is H or
--C.sub.1-3alkyl; each R.sup.6 is independently halo,
--C.sup.1-4alkyl, --C.sub.0-3alkylOR.sup.7,
--C.sub.0-3alkylNR.sup.9R.sup.10,
--C.sub.0-3alkyl-CONR.sup.9R.sup.10, --CN, oxo,
--SO.sub.2--C.sub.1-3alkyl, or --SO.sub.2NR.sup.9R.sup.10; R.sup.7
and R.sup.8 are each independently selected from --H,
--C.sub.1-3alkyl, and --C.sub.2-4alkylOC.sub.0-3alkyl; R.sup.9 and
R.sup.10 are each independently selected from --H and
--C.sub.1-3alkyl; or R.sup.9 and R.sup.10 may join together with
the nitrogen to which they are attached, to form a 4 to 7-membered
heterocyclyl optionally substituted by one or two substituents
independently selected from --C.sub.1-3alkyl optionally substituted
with up to 3 fluorine atoms, --C.sub.2-4alkylOH, --OH, and F; m is
an integer selected from 2, 3, or 4; and n is an integer selected
from 0, 1, 2, 3, or 4.
32. The compound or pharmaceutically acceptable salt thereof
according to claim 31 wherein R.sup.1 is methyl.
33. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein R.sup.2 is methyl, ethyl, propyl,
iso-propyl, butyl, --CH.sub.2CH.sub.2CH(CH.sub.3).sub.2,
--CH.sub.2CH(CH.sub.3).sub.2, --CH.sub.2CH.sub.2OR.sup.7,
--CH.sub.2CH.sub.2CH.sub.2OR.sup.7, --CH.sub.2CH(CH.sub.3)OR.sup.7,
--CH.sub.2CH.sub.2CH(CH.sub.3)OR.sup.7,
CH.sub.2CH.sub.2CH(OR.sup.7).sub.2,
--CH.sub.2CH.sub.2CH(CH.sub.3)NR.sup.7R.sup.8,
--CH.sub.2CH.sub.2CH.sub.2NR.sup.7R.sup.8,
--(CH.sub.2).sub.mSO.sub.2CH.sub.3,
--(CH.sub.2).sub.mC(O)NHCH.sub.3, --(CH.sub.2).sub.mCN,
--(CH.sub.2).sub.mCO.sub.2R.sup.7, --(CH.sub.2).sub.mCF.sub.3, and
--(CH.sub.2).sub.mNHCO.sub.2C(CH.sub.3).sub.3.
34. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein R.sup.2 is
--(CH.sub.2).sub.nC.sub.5-6heteroaryl wherein the
C.sub.5-6heteroaryl is selected from furanyl, thienyl, pyrrolyl,
triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl,
pyridinyl, pyridazinyl, pyrazinyl, and pyrimidinyl, wherein said
furanyl, thienyl, pyrrolyl, triazolyl, thiazolyl, oxazolyl,
isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, triazolyl,
tetrazolyl, isoxazolyl, pyridinyl, pyridazinyl, pyrazinyl, or
pyrimidinyl is optionally substituted by one or two substituents
independently selected from halo, C.sub.1-4alkyl,
C.sub.3-4cycloalkyl, and --C.sub.0-3alkylOR.sup.5.
35. The compound or pharmaceutically acceptable salt thereof
according to claim 34, wherein the C.sub.5-6 heteroaryl is
pyrazolyl optionally substituted by C.sub.1-4alkyl or
--C.sub.0-3alkylOR.sup.5.
36. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein R.sup.3 is --H, methyl, fluoro,
--OCH.sub.3, or --OH.
37. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein R.sup.4 is phenyl optionally
substituted by one, two, or three R.sup.6 groups which may be the
same or different.
38. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein R.sup.4 is a heteroaryl group which
is indolyl optionally substituted by one, two, or three R.sup.6
groups which may be the same or different.
39. The compound or pharmaceutically acceptable salt thereof
according to claim 38, wherein R.sup.4 is 1H-indol-4-yl.
40. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein n is 0 or 2.
41. The compound or pharmaceutically acceptable salt thereof
according to claim 31, wherein m is 2.
42. A compound which is selected from
6-Benzyl-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyrazol-4-yl)pyridine-2,4-di-
carboxamide;
6-Benzyl-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,4-dicarboxami-
de;
(S)-N.sup.2-Methyl-6-(1-phenylethyl)-N.sup.4-(1H-pyrazol-4-yl)pyridine-
-2,4-dicarboxamide; and
(S)-6-(Methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyr-
idine-2,4-dicarboxamide; or a pharmaceutically acceptable salt
thereof.
43. A pharmaceutical composition comprising a compound or a
pharmaceutically acceptable salt thereof according to claim 31 and
one or more pharmaceutically acceptable excipients.
44. A combination comprising a compound or a pharmaceutically
acceptable salt thereof according to claim 31 together with one or
more other therapeutically active agents.
45. A method of treatment of a disease or condition in a human for
which a bromodomain inhibitor is indicated, the method comprising
administering to the human in need thereof a therapeutically
effective amount of the compound or pharmaceutically acceptable
salt thereof as according to claim 31.
46. The method of treatment according to claim 45, wherein the
disease or condition is an acute or chronic autoimmune and/or
inflammatory condition.
47. The method of treatment according to claim 45, wherein the
disease or condition involves an inflammatory response to an
infection with bacteria, a virus, fungi, a parasite, or their
toxins.
48. The method of treatment according to claim 45, wherein the
disease or condition is a viral infection.
49. The method of treatment according to claim 44, wherein the
disease or condition is cancer.
50. The method of treatment according to claim 45, wherein the
disease or condition is rheumatoid arthritis.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to pyridyl derivatives
which are bromodomain inhibitors, pharmaceutical compositions
comprising the compounds and the use of the compounds or the
compositions in the treatment of various diseases or conditions,
for example acute or chronic autoimmune and/or inflammatory
conditions, viral infections and cancer.
BACKGROUND TO THE INVENTION
[0002] The genomes of eukaryotic organisms are highly organised
within the nucleus of the cell. The long strands of duplex DNA are
wrapped around an octomer of histone proteins (most usually
comprising two copies of histones H2A, H2B, H3 and H4) to form a
nucleosome. This basic unit is then further compressed by the
aggregation and folding of nucleosomes to form a highly condensed
chromatin structure. A range of different states of condensation
are possible, and the tightness of this structure varies during the
cell cycle, being most compact during the process of cell division.
Chromatin structure plays a critical role in regulating gene
transcription, which cannot occur efficiently from highly condensed
chromatin. The chromatin structure is controlled by a series of
post translational modifications to histone proteins, notably
histones H3 and H4, and most commonly within the histone tails
which extend beyond the core nucleosome structure. These
modifications include acetylation, methylation, phosphorylation,
ubiquitinylation and SUMOylation. These epigenetic marks are
written and erased by specific enzymes, which place the tags on
specific residues within the histone tail, thereby forming an
epigenetic code, which is then interpreted by the cell to allow
gene specific regulation of chromatin structure and thereby
transcription.
[0003] Histone acetylation is most usually associated with the
activation of gene transcription, as the modification loosens the
interaction of the DNA and the histone octomer by changing the
electrostatics. In addition to this physical change, specific
proteins recognise and bind to acetylated lysine residues within
histones to read the epigenetic code. Bromodomains are small
(.about.110 amino acid) distinct domains within proteins that bind
to acetylated lysine resides commonly but not exclusively in the
context of histones. There is a family of around 50 proteins known
to contain bromodomains, and they have a range of functions within
the cell.
[0004] The BET family of bromodomain containing proteins comprises
4 proteins (BRD2, BRD3, BRD4 and BRDT) which contain tandem
bromodomains capable of binding to two acetylated lysine residues
in close proximity, increasing the specificity of the interaction.
Numbering from the N-terminal end of each BET protein the tandem
bromodomains are typically labelled Binding Domain 1 (BD1) and
Binding Domain 2 (BD2) (Chung et al., J Med. Chem., 2011, 54,
3827-3838).
[0005] Chan et al. report that BET bromodomain inhibition
suppresses transcriptional responses to cytokine-Jak-STAT
signalling in a gene-specific maner in human monocytes, which
suggests that BET inhibition reduces inflammation partially through
suppression of cytokine activity. (Chan et al., Eur. J. Immunol.,
2015, 45: 287-297).
[0006] Klein et al. report that the bromodomain protein inhibitor
I-BET151 suppresses expression of inflammatory genes and matrix
degrading enzymes in rheumatoid arthritis synovial fibroblasts,
which suggests a therapeutic potential in the targeting of
epigenetic reader proteins in rheumatoid arthritis. (Klein et al.,
Ann. Rheum. Dis., 2014, 0:1-8).
[0007] Park-Min et al. report that I-BET151 that targets bromo and
extra-terminal (BET) proteins that `read` chromatin states by
binding to acetylated histones, strongly suppresses
osteoclastogenesis. (Park-Min et al. Nature Communications, 2014,
5, 5418).
[0008] PCT patent applications PCT/EP2016/070519, PCT/EP2016/072216
and PCT/EP2016/073532 each describe a series of pyridone
derivatives as bromodomain inhibitors.
SUMMARY OF THE INVENTION
[0009] The invention is directed to compounds of formula (I)
##STR00001##
or a salt thereof [0010] wherein:
[0011] R.sup.1 is --C.sub.1-3alkyl or cyclopropyl;
[0012] R.sup.2 is H, --CH.sub.3, C.sub.2-6alkyl optionally
substituted by one, two, three, four or five fluoro,
--C.sub.2-6alkylOR.sup.7, --C.sub.2-6alkylNR.sup.7R.sup.8,
--(CH.sub.2).sub.mSO.sub.2C.sub.1-3alkyl,
--(CH.sub.2).sub.mC(O)NR.sup.7R.sup.8, --(CH.sub.2).sub.mCN,
--(CH.sub.2).sub.mCO.sub.2R.sup.7,
--(CH.sub.2).sub.mNHCO.sub.2C(CH.sub.3).sub.3; or
[0013] R.sup.2 is --(CH.sub.2).sub.nC.sub.5-6heteroaryl wherein
C.sub.5-6heteroaryl is optionally substituted by one or two
substituents independently selected from halo, --C.sub.1-4alkyl,
--C.sub.3-4cycloalkyl and --C.sub.0-4alkylOR.sup.5;
[0014] R.sup.3 is H, --C.sub.1-4alkyl, cyclopropyl, fluoro, chloro,
--CH.sub.2F, --C.sub.0-3alkylOR.sup.5 or --C.sub.0-3alkylCN;
[0015] R.sup.4 is phenyl or a heteroaryl group wherein each are
optionally substituted by one, two or three R.sup.6 groups which
may be the same or different;
[0016] R.sup.5 is H or --C.sub.1-3alkyl;
[0017] each R.sup.6 is independently halo, --C.sub.1-4alkyl,
--C.sub.0-3alkylOR.sup.7, --C.sub.0-3alkylNR.sup.9R.sup.10,
--C.sub.0-3alkyl-CONR.sup.9R.sup.10, --CN, oxo,
--SO.sub.2--C.sub.1-3alkyl or --SO.sub.2NR.sup.9R.sup.10;
[0018] R.sup.7 and R.sup.8 are each independently selected from
--H, --C.sub.1-3alkyl and --C.sub.2-4alkylOC.sub.0-3alkyl;
[0019] R.sup.9 and R.sup.10 are each independently selected from
--H and --C.sub.1-3alkyl; or R.sup.9 and R.sup.10 may join together
with the nitrogen to which they are attached, to form a 4 to
7-membered heterocyclyl optionally substituted by one or two
substituents independently selected from --C.sub.1-3alkyl
optionally substituted with up to 3 fluorine atoms,
--C.sub.2-4alkylOH, --OH and F;
[0020] m is an integer selected from 2, 3 or 4; and
[0021] n is an integer selected from 0, 1, 2, 3 or 4.
[0022] Compounds of the invention have been shown to be bromodomain
inhibitors, in particular BD2 selective and may be useful in the
treatment of various diseases or conditions, for example acute or
chronic auto-immune and/or inflammatory conditions, for example
rheumatoid arthritis and cancer. Accordingly, the invention is
further directed to pharmaceutical compositions comprising a
compound of formula (I), or a pharmaceutically acceptable salt
thereof. The invention is still further directed to methods of
treatment of diseases or conditions associated with bromodomains
using a compound of formula (I) or a pharmaceutically acceptable
salt thereof, or a pharmaceutical composition comprising a compound
of formula (I) or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Compounds of formula (I) and salts thereof are referred to
herein as "compounds of the invention".
[0024] "BD2" refers to Binding Domain 2 of any of the the BET
family of proteins BRD2, BRD3, BRD4 or BRDT.
[0025] "Alkyl" refers to a saturated hydrocarbon chain having the
specified number of carbon atoms. For example, the term
"C.sub.1-3alkyl" or "C.sub.1-4alkyl" as used herein refers to a
straight or branched alkyl group having from 1 to 3 carbon atoms or
1 to 4 carbon atoms respectively. Further, the term
"C.sub.0-3alkyl" refers to a straight or branched alkyl group
having from 0 (i.e. a bond) to 3 carbon atoms. Representative
branched alkyl groups have one, two or three branches. An alkyl
group may form part of a chain, for example,
--C.sub.0-3alkylOR.sup.5 refers to a straight or branched alkyl
chain having from 0 (i.e. a bond) to 3 carbon atoms linked to a
group R.sup.5. "Alkyl" includes, but is not limited to, methyl,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, pentyl
and hexyl.
[0026] "Halo" refers to a halogen radical, for example, fluoro,
chloro, bromo, or iodo.
[0027] "Heteroaryl" refers to a monocyclic or bicyclic group having
5, 6, 8, 9, 10 or 11 member atoms, including 1, 2 or 3 heteroatoms
independently selected from nitrogen, sulphur and oxygen, wherein
at least a portion of the group is aromatic. The point of
attachment to the rest of the molecule may be by any suitable
carbon or nitrogen atom. Examples of "heteroaryl" groups include,
but are not limited to, furanyl, thienyl, pyrrolyl, imidazolyl,
pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl,
oxadiazolyl, thiadiazolyl, isothiazolyl, pyridinyl, pyridazinyl,
pyrazinyl, pyrimidinyl, triazinyl, benzofuranyl, isobenzofuryl,
2,3-dihydrobenzofuryl, 1,3-benzodioxolyl, dihydrobenzodioxinyl,
benzothienyl, benzazepinyl, 2,3,4,5-tetrahydro-1H-benzo[d]azepinyl,
indolizinyl, indolyl, indolinyl, isoindolyl, dihydroindolyl,
benzimidazolyl, dihydrobenzimidazolyl, benzoxazolyl,
dihydrobenzoxazolyl, benzthiazolyl, benzoisothiazolyl,
dihydrobenzoisothiazolyl, indazolyl, imidazopyridinyl,
pyrazolopyridinyl, pyrrolopyridinyl, benzotriazolyl,
triazolopyridinyl, purinyl, quinolinyl, tetrahydroquinolinyl,
isoquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, cinnolinyl,
phthalazinyl, quinazolinyl, 1,5-naphthyridinyl, 1,6-naphthyridinyl,
1,7-naphthyridinyl, 1,8-naphthyridinyl, and pteridinyl.
[0028] "C.sub.5-6heteroaryl" refers to a monocyclic aromatic group
having 5 or 6 member atoms, including 1, 2, 3 or 4 heteroatoms
independently selected from nitrogen, sulphur and oxygen. The point
of attachment to the rest of the molecule may be by any suitable
carbon or nitrogen atom. Examples of "C.sub.5-6heteroaryl" groups
include, but are not limited to, furanyl, thienyl, pyrrolyl,
triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl,
pyridinyl, pyridazinyl, pyrazinyl and pyrimidinyl.
[0029] "Heteroatom" refers to a nitrogen, sulfur, or oxygen
atom.
[0030] "4 to 7-membered heterocyclyl" refers to a non-aromatic
heterocyclic ring system containing 4, 5, 6 or 7 ring member atoms,
including one heteroatom and optionally containing a further
heteroatom selected from nitrogen, oxygen or sulphur. Examples of
"4 to 7-membered heterocyclyl" groups include, but are not limited
to, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl and
morpholinyl.
[0031] "Member atoms" refers to the atom or atoms that form a chain
or ring. Where more than one member atom is present in a chain and
within a ring, each member atom is covalently bound to an adjacent
member atom in the chain or ring. Atoms that make up a substituent
group attached to a chain or ring are not member atoms in the chain
or ring.
[0032] "Substituted" in reference to a group indicates that a
hydrogen atom attached to a member atom within a group is replaced.
It should be understood that the term "substituted" includes the
implicit provision that such substitution be in accordance with the
permitted valence of the substituted atom and the substituent and
that the substitution results in a stable compound (i.e. one that
does not spontaneously undergo transformation such as
rearrangement, cyclisation, or elimination). In certain
embodiments, a single atom may be substituted with more than one
substituent as long as such substitution is in accordance with the
permitted valence of the atom. Suitable substituents are defined
herein for each substituted or optionally substituted group.
"Pharmaceutically acceptable" refers to those compounds, materials,
compositions, and dosage forms which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
human beings and animals without excessive toxicity, irritation, or
other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0033] "Pharmaceutically acceptable excipient" refers to a
pharmaceutically acceptable material, composition or vehicle
involved in giving form or consistency to the pharmaceutical
composition. Each excipient must be compatible with the other
ingredients of the pharmaceutical composition when commingled such
that interactions which would substantially reduce the efficacy of
the compound of formula (I) or a pharmaceutically acceptable salt
thereof when administered to a patient are avoided. In addition,
each excipient must of course be pharmaceutically acceptable e.g.
of sufficiently high purity.
[0034] "rac"refers to the racemic mixture of the compounds of
formula (I).
[0035] The compounds of the invention may exist in solid or liquid
form. In the solid state, the compounds of the invention may exist
in crystalline or non-crystalline form, or as a mixture thereof.
For compounds of the invention that are in crystalline form, the
skilled artisan will appreciate that pharmaceutically acceptable
solvates may be formed wherein solvent molecules are incorporated
into the crystalline lattice during crystallization. Solvates may
involve non-aqueous solvents such as ethanol, iso-propyl alcohol,
dimethylsulfoxide (DMSO), acetic acid, ethanolamine, and ethyl
acetate, or they may involve water as the solvent that is
incorporated into the crystalline lattice. Solvates wherein water
is the solvent that is incorporated into the crystalline lattice
are typically referred to as "hydrates". Hydrates include
stoichiometric hydrates as well as compositions containing variable
amounts of water. The invention includes all such solvates.
[0036] It will be further appreciated that certain compounds of the
invention that exist in crystalline form, including the various
solvates thereof, may exhibit polymorphism (i.e. the capacity to
occur in different crystalline structures). These different
crystalline forms are typically known as "polymorphs". The
invention includes such polymorphs. Polymorphs have the same
chemical composition but differ in packing, geometrical
arrangement, and other descriptive properties of the crystalline
solid state. Polymorphs, therefore, may have different physical
properties such as shape, density, hardness, deformability,
stability, and dissolution properties. Polymorphs typically exhibit
different melting points, IR spectra, and X-ray powder diffraction
patterns, which may be used for identification. It will be
appreciated that different polymorphs may be produced, for example,
by changing or adjusting the reaction conditions or reagents, used
in making the compound. For example, changes in temperature,
pressure, or solvent may result in polymorphs. In addition, one
polymorph may spontaneously convert to another polymorph under
certain conditions. Polymorphic forms of compounds of formula (I)
may be characterized and differentiated using a number of
conventional analytical techniques, including, but not limited to,
X-ray powder diffraction (XRPD) patterns, infrared (IR) spectra,
Raman spectra, differential scanning calorimetry (DSC),
thermogravimetric analysis (TGA) and solid state nuclear magnetic
resonance (SSNMR).
[0037] The compounds according to formula (I) may contain one or
more asymmetric centres (also referred to as a chiral centres) and
may, therefore, exist as individual enantiomers, diastereoisomers,
or other stereoisomeric forms, or as mixtures thereof. Chiral
centres, such as chiral carbon atoms, may also be present in a
substituent such as an alkyl group. Where the stereochemistry of a
chiral centre present in formula (I), or in any chemical structure
illustrated herein, is not specified, the structure is intended to
encompass any stereoisomer and all mixtures thereof. Thus,
compounds according to formula (I) containing one or more chiral
centres may be used as racemic mixtures, enantiomerically-enriched
mixtures, or as enantiomerically-pure individual stereoisomers.
Accordingly, the present invention encompasses all isomers of the
compounds of formula (I) whether as individual isomers isolated
such as to be substantially free of the other isomer (i.e. pure) or
as mixtures (i.e. racemic mixtures). An individual isomer isolated
such as to be substantially free of the other isomer (i.e. pure)
may be isolated such that less than 10%, particularly less than
about 1%, for example less than about 0.1% of the other isomer is
present.
[0038] Racemic compounds with a single stereocentre are denoted
with either no stereochemistry (single bond) or have the annotation
(+/-) or rac. Racemic compounds with two or more stereocentres
where relative stereochemistry is known are denoted cis or trans as
drawn in the structure. Resolved single enantiomers with unknown
absolute stereochemistry but known relative stereochemistry are
referred to with (R* or S*) with the appropriate relative
stereochemistry depicted.
[0039] Where diastereoisomers are represented and only the relative
stereochemistry is referred to, the bold or hashed solid bond
symbols (/) are used. Where the absolute stereochemistry is known
and the compound is a single enantiomer, the bold or hashed wedges
symbols (/) are used as appropriate.
[0040] Individual stereoisomers of a compound according to formula
(I) which contain one or more asymmetric centres may be resolved by
methods known to those skilled in the art. For example, such
resolution may be carried out (1) by formation of diastereoisomeric
salts, complexes or other derivatives; (2) by selective reaction
with a stereoisomer-specific reagent, for example by enzymatic
oxidation or reduction; or (3) by gas-liquid or liquid
chromatography in a chiral environment, for example, on a chiral
support such as silica with a bound chiral ligand or in the
presence of a chiral solvent. It will be appreciated that where the
desired stereoisomer is converted into another chemical entity by
one of the separation procedures described above, a further step is
required to liberate the desired form. Alternatively, specific
stereoisomers may be synthesised by asymmetric synthesis using
optically active reagents, substrates, catalysts or solvents, or by
converting one enantiomer to the other by asymmetric
transformation.
[0041] It will be appreciated that, for compounds of formula (I)
tautomers may be observed. Any comment relating to the biological
activity of a tautomer should be taken to include both
tautomers.
[0042] It is to be understood that the references herein to
compounds of formula (I) and salts thereof covers the compounds of
formula (I) as free bases, or as salts thereof, for example as
pharmaceutically acceptable salts thereof. Thus, in one embodiment,
the invention is directed to compounds of formula (I) as the free
base. In another embodiment, the invention is directed to compounds
of formula (I) and salts thereof. In a further embodiment, the
invention is directed to compounds of formula (I) and
pharmaceutically acceptable salts thereof.
[0043] Because of their potential use in medicine, salts of the
compounds of formula (I) are desirably pharmaceutically acceptable.
Suitable pharmaceutically acceptable salts can include acid
addition salts or base addition salts. For a review of suitable
pharmaceutically acceptable salts see Berge et al., J. Pharm. Sci.,
66:1-19, (1977). Typically, a pharmaceutically acceptable salt may
be readily prepared by using a desired acid or base as appropriate.
The resultant salt may precipitate from solution and be collected
by filtration or may be recovered by evaporation of the
solvent.
[0044] A pharmaceutically acceptable acid addition salt can be
formed by reaction of a compound of formula (I) with a suitable
inorganic or organic acid (such as hydrobromic, hydrochloric,
sulphuric, nitric, phosphoric, succinic, maleic, acetic, propionic,
fumaric, citric, tartaric, lactic, benzoic, salicylic, aspartic,
p-toluenesulphonic, benzenesulphonic, methanesulphonic,
ethanesulphonic, naphthalenesulphonic such as
2-naphthalenesulphonic, or hexanoic acid), optionally in a suitable
solvent such as an organic solvent, to give the salt which is
usually isolated for example by crystallisation and filtration or
by evaporation followed by trituration. A pharmaceutically
acceptable acid addition salt of a compound of formula (I) can
comprise or be for example a hydrobromide, hydrochloride, sulfate,
nitrate, phosphate, succinate, maleate, acetate, propionate,
fumarate, citrate, tartrate, lactate, benzoate, salicylate,
glutamate, aspartate, p-toluenesulphonate, benzenesulphonate,
methanesulphonate, ethanesulphonate, naphthalenesulphonate (e.g.
2-naphthalenesulphonate) or hexanoate salt.
[0045] Other non-pharmaceutically acceptable salts, e.g. formates
or trifluoroacetates, may be used, for example in the isolation of
the compounds of formula (I), and are included within the scope of
this invention.
[0046] The invention includes within its scope all possible
stoichiometric and non-stoichiometric forms of the salts of the
compounds of formula (I).
[0047] It will be appreciated from the foregoing that included
within the scope of the invention are solvates, isomers and
polymorphic forms of the compounds of formula (I) and salts
thereof.
STATEMENT OF THE INVENTION
[0048] In a first aspect there are provided compounds of formula
(I):
##STR00002##
or a salt thereof
[0049] R.sup.1 is --C.sub.1-3alkyl or cyclopropyl;
[0050] R.sup.2 is --H, --CH.sub.3, C.sub.2-6alkyl optionally
substituted by up to five fluoro, --C.sub.2-6alkylOR.sup.7,
--C.sub.2-6alkylINR.sup.7R.sup.8,
--(CH.sub.2).sub.mSO.sub.2C.sub.1-3alkyl,
--(CH.sub.2).sub.mC(O)NR.sup.7R.sup.8, --(CH.sub.2).sub.mCN,
--(CH.sub.2).sub.mCO.sub.2R.sup.7,
--(CH.sub.2).sub.mNHCO.sub.2C(CH.sub.3).sub.3; or
[0051] R.sup.2 is --(CH.sub.2).sub.nC.sub.5-6heteroaryl wherein
C.sub.5-6heteroaryl is optionally substituted by one or two
substituents independently selected from halo, --C.sub.1-4alkyl,
--C.sub.3-4cycloalkyl and --C.sub.0-4alkylOR.sup.5;
[0052] R.sup.3 is --H, --C.sub.1-4alkyl, cyclopropyl, fluoro,
chloro, --CH.sub.2F, C.sub.0-3alkylOR.sup.5 or
--C.sub.0-3alkylCN;
[0053] R.sup.4 is phenyl or a heteroaryl group wherein each are
optionally substituted by one, two or three R.sup.6 groups which
may be the same or different;
[0054] R.sup.5 is --H or --C.sub.1-3alkyl;
[0055] each R.sup.6 is independently halo, --C.sub.1-4alkyl,
--C.sub.0-3alkylOR.sup.7, --C.sub.0-3alkylNR.sup.9R.sup.10,
--C.sub.0-3alkyl-CONR.sup.9R.sup.10, --CN, oxo,
--SO.sub.2--C.sub.1-3alkyl or --SO.sub.2NR.sup.9R.sup.10;
[0056] R.sup.7 and R.sup.8 are each independently selected from
--H, --C.sub.1-3alkyl and --C.sub.2-4alkylOC.sub.0-3alkyl;
[0057] R.sup.9 and R.sup.10 are each independently selected from
--H and --C.sub.1-3alkyl; or R.sup.9 and R.sup.10 may join together
with the nitrogen to which they are attached, to form a 4 to
7-membered heterocyclyl optionally substituted by one or two
substituents independently selected from --C.sub.1-3alkyl
optionally substituted with up to 3 fluorine atoms,
--C.sub.2-4alkylOH, --OH and F;
[0058] m is an integer selected from 2, 3 or 4; and
[0059] n is an integer selected from 0, 1, 2, 3 or 4.
[0060] In one embodiment R.sup.1 is methyl.
[0061] In one embodiment R.sup.2 is H.
[0062] In one embodiment R.sup.2 is selected from methyl, ethyl,
propyl, iso-propyl, butyl, --CH.sub.2CH.sub.2CH(CH.sub.3).sub.2,
--CH.sub.2CH(CH.sub.3).sub.2, --CH.sub.2CH.sub.2OR.sup.7,
--CH.sub.2CH.sub.2CH.sub.2OR.sup.7, --CH.sub.2CH(CH.sub.3)OR.sup.7,
--CH.sub.2CH.sub.2CH(CH.sub.3)OR.sup.7,
--CH.sub.2CH.sub.2CH(OR.sup.7).sub.2,
--CH.sub.2CH.sub.2CH(CH.sub.3)NR.sup.7R.sup.8,
--CH.sub.2CH.sub.2CH.sub.2NR.sup.7R.sup.8,
--(CH.sub.2).sub.mSO.sub.2CH.sub.3,
--(CH.sub.2).sub.mC(O)NHCH.sub.3, --(CH.sub.2).sub.mCN,
--(CH.sub.2).sub.mCO.sub.2R.sup.7, --(CH.sub.2).sub.mCF.sub.3 and
--(CH.sub.2).sub.mNHCO.sub.2C(CH.sub.3).sub.3.
[0063] In another embodiment R.sup.2 is --C.sub.1-6alkyl selected
from methyl, ethyl, propyl, iso-propyl, butyl,
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2 and
--CH.sub.2CH(CH.sub.3).sub.2. In another embodiment R.sup.2 is
--C.sub.1-6alkylOR.sup.7 selected from --CH.sub.2CH.sub.2OR.sup.7,
--CH.sub.2CH.sub.2CH.sub.2OR.sup.7, --CH.sub.2CH(CH.sub.3)OR.sup.7,
--CH.sub.2CH.sub.2CH(CH.sub.3)OR.sup.7 and
--CH.sub.2CH.sub.2CH(OR.sup.7).sub.2. In another embodiment R.sup.2
is --C.sub.1-6alkylNR.sup.7R.sup.8 selected from
--CH.sub.2CH.sub.2CH(CH.sub.3)NR.sup.7R.sup.8 and
--CH.sub.2CH.sub.2CH.sub.2NR.sup.7R.sup.8. In another embodiment
R.sup.2 is --(CH.sub.2).sub.mSO.sub.2CH.sub.3. In another
embodiment R.sup.2 is --(CH.sub.2).sub.mC(O)NHCH.sub.3. In another
embodiment R.sup.2 is --(CH.sub.2).sub.mCN. In another embodiment
R.sup.2 is --(CH.sub.2).sub.mCO.sub.2R.sup.7. In another embodiment
R.sup.2 is --(CH.sub.2).sub.mCF.sub.3. In another embodiment
R.sup.2 is --(CH.sub.2).sub.mNHCO.sub.2C(CH.sub.3).sub.3.
[0064] In another embodiment R.sup.2 is
--(CH.sub.2).sub.nC.sub.5-6heteroaryl wherein the
C.sub.5-6heteroaryl is selected from furanyl, thienyl, pyrrolyl,
triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl,
imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl,
pyridinyl, pyridazinyl, pyrazinyl and pyrimidinyl said groups being
optionally substituted by one or two substituents independently
selected from halo, C.sub.1-4alkyl (such as methyl),
C.sub.3-4cycloalkyl and --C.sub.0-3alkylOR.sup.5.
[0065] In another embodiment there is provided compounds of formula
(I) in which R.sup.2 is --(CH.sub.2).sub.nC.sub.5-6heteroaryl
wherein the C.sub.5-6heteroaryl is pyrazolyl optionally substituted
by C.sub.1-4alkyl or --C.sub.0-3alkylOR.sup.5. In a particular
embodiment there is provided compounds of formula (I) in which
R.sup.2 is --(CH.sub.2).sub.nC.sub.5-6heteroaryl wherein the
C.sub.5-6heteroaryl is selected from the group consisting of
##STR00003##
wherein * denotes the point of attachment to the alkyl residue.
[0066] In one embodiment R.sup.3 is --H, methyl, fluoro,
--OCH.sub.3 or --OH.
[0067] In one embodiment R.sup.4 is phenyl optionally substituted
by one, two or three R.sup.6 groups which may be the same or
different. In another embodiment R.sup.4 is unsubstituted
phenyl.
[0068] In another embodiment R.sup.4 is a heteroaryl group which is
indolyl (e.g 1H-indol-4-yl) optionally substituted by one, two or
three R.sup.6 groups which may be the same or different. In another
embodiment R.sup.4 is a heteroaryl group which is
1H-indol-4-yl.
[0069] In one embodiment each R.sup.6 is independently halo,
--C.sub.1-4alkyl or --C.sub.0-3alkylOR.sup.7.
[0070] In one embodiment m is 2 or 3.
[0071] In one embodiment n is 0, 1 or 2. In one embodiment n is 0
or 2. In a further embodiment n is 0. In a yet further embodiment n
is 2.
[0072] It is to be understood that the present invention covers all
combinations of substituent groups described hereinabove.
[0073] Compounds of the invention include the compounds of Examples
1 to 81 and salts thereof.
[0074] In one embodiment the compound of formula (I) is selected
from: [0075]
6-Benzyl-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyrazol-4-yl)pyridine-
-2,4-dicarboxamide; [0076]
6-Benzyl-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,4-dicarboxami-
de; [0077]
(S)-N.sup.2-Methyl-6-(1-phenylethyl)-N.sup.4-(1pyrazol-4-yl)pyr-
idine-2,4-dicarboxamide; and [0078]
(S)-6-(Methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyr-
idine-2,4-dicarboxamide [0079] or a salt thereof.
[0080] In a second aspect of the present invention, there is
provided a pharmaceutical composition comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof and one
or more pharmaceutically acceptable excipients.
[0081] In a third aspect of the present invention, there is
provided a compound of formula (I), or a pharmaceutically
acceptable salt thereof for use in therapy, in particular in the
treatment of diseases or conditions for which a bromodomain
inhibitor is indicated.
[0082] In a fourth aspect of the present invention, there is
provided a method of treating diseases or conditions for which a
bromodomain inhibitor is indicated in a subject (e.g. a human
subject) in need thereof which comprises administering a
therapeutically effective amount of a compound of formula (I) or a
pharmaceutically acceptable salt thereof.
[0083] In a fifth aspect of the present invention, there is
provided the use of a compound of formula (I), or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament for the treatment of diseases or conditions for which a
bromodomain inhibitor is indicated.
Statement of Use
[0084] The compounds of formula (I) and salts thereof are
bromodomain inhibitors, and thus are believed to have potential
utility in the treatment of diseases or conditions for which a
bromodomain inhibitor is indicated.
[0085] Bromodomain inhibitors are believed to be useful in the
treatment of a variety of diseases or conditions related to
systemic or tissue inflammation, inflammatory responses to
infection or hypoxia, cellular activation and proliferation, lipid
metabolism, fibrosis and in the prevention and treatment of viral
infections.
[0086] Bromodomain inhibitors may be useful in the treatment of a
wide variety of acute or chronic autoimmune and/or inflammatory
conditions such as rheumatoid arthritis, psoriatic arthritis,
ankylosing spondylitis, osteoarthritis, acute gout, psoriasis,
systemic lupus erythematosus, multiple sclerosis, inflammatory
bowel disease (Crohn's disease and ulcerative colitis), asthma,
chronic obstructive airways disease, pneumonitis, myocarditis,
pericarditis, myositis, eczema, dermatitis (including atopic
dermatitis), alopecia, vitiligo, bullous skin diseases, nephritis,
vasculitis, hypercholesterolemia, atherosclerosis, Alzheimer's
disease, Sjogren's syndrome, sialoadenitis, central retinal vein
occlusion, branched retinal vein occlusion, Irvine-Gass syndrome
(post cataract and post-surgical), retinitis pigmentosa, pars
planitis, birdshot retinochoroidopathy, epiretinal membrane, cystic
macular edema, parafoveal telengiectasis, tractional maculopathies,
vitreomacular traction syndromes, retinal detachment,
neuroretinitis, idiopathic macular edema, retinitis, dry eye
(keratoconjunctivitis Sicca), vernal keratoconjunctivitis, atopic
keratoconjunctivitis, uveitis (such as anterior uveitis, pan
uveitis, posterior uveitis, uveitis-associated macular edema),
scleritis, diabetic retinopathy, diabetic macula edema, age-related
macular dystrophy, hepatitis, pancreatitis, primary biliary
cirrhosis, sclerosing cholangitis, acute alcoholic hepatitis,
chronic alcoholic hepatitis, alcoholic steato-hepatitis,
non-alcoholic steato-hepatitis (NASH), cirrhosis, Childs-Pugh
cirrhosis, autoimmune hepatitis, fulminant hepatitis, chronic viral
hepatitis, alcoholic liver disease, systemic sclerosis, systemic
sclerosis with associated interstitial lung disease, sarcoidosis,
neurosarcoidosis, Addison's disease, hypophysitis, thyroiditis,
Type I diabetes, Type II diabetes, giant cell arteritis, nephritis
including lupus nephritis, vasculitis with organ involvement such
as glomerulonephritis, vasculitis including giant cell arteritis,
Wegener's granulomatosis, Polyarteritis nodosa, Behcet's disease,
Kawasaki disease, Takayasu's Arteritis, pyoderma gangrenosum,
vasculitis with organ involvement, acute rejection of transplanted
organs and systemic sclerosis.
[0087] In one embodiment the acute or chronic autoimmune and/or
inflammatory condition is a disorder of lipid metabolism mediated
via the regulation of APO-A1 such as hypercholesterolemia,
atherosclerosis or Alzheimer's disease.
[0088] In another embodiment the acute or chronic autoimmune and/or
inflammatory condition is a respiratory disorder such as asthma or
chronic obstructive airways disease.
[0089] In another embodiment the acute or chronic autoimmune and/or
inflammatory condition is a systemic inflammatory disorder such as
rheumatoid arthritis, osteoarthritis, acute gout, psoriasis,
systemic lupus erythematosus, multiple sclerosis or inflammatory
bowel disease (Crohn's disease or Ulcerative colitis).
[0090] In another embodiment, the acute or chronic autoimmune
and/or inflammatory condition is multiple sclerosis.
[0091] In another embodiment, the acute or chronic autoimmune
and/or inflammatory condition is Type I diabetes.
[0092] In another embodiment, the acute or chronic autoimmune
and/or inflammatory condition is rheumatoid arthritis.
[0093] Bromodomain inhibitors may be useful in the treatment of
depression.
[0094] Bromodomain inhibitors may be useful in the treatment of
diseases or conditions which involve inflammatory responses to
infections with bacteria, viruses, fungi, parasites or their
toxins, such as sepsis, acute sepsis, sepsis syndrome, septic
shock, endotoxaemia, systemic inflammatory response syndrome
(SIRS), multi-organ dysfunction syndrome, toxic shock syndrome,
acute lung injury, ARDS (adult respiratory distress syndrome),
acute renal failure, fulminant hepatitis, burns, acute
pancreatitis, post-surgical syndromes, sarcoidosis, Herxheimer
reactions, encephalitis, myelitis, meningitis, malaria and SIRS
associated with viral infections such as influenza, herpes zoster,
herpes simplex and coronavirus. In one embodiment the disease or
condition which involves an inflammatory response to an infection
with bacteria, a virus, fungi, a parasite or their toxins is acute
sepsis.
[0095] Bromodomain inhibitors may be useful in the treatment of
conditions associated with ischaemia-reperfusion injury such as
myocardial infarction, cerebro-vascular ischaemia (stroke), acute
coronary syndromes, renal reperfusion injury, organ
transplantation, coronary artery bypass grafting, cardio-pulmonary
bypass procedures, pulmonary, renal, hepatic, gastro-intestinal or
peripheral limb embolism.
[0096] Bromodomain inhibitors may be useful in the treatment of
cardiovascular diseases such as coronary artery diseases (for
example, angina or myocardial infarction), pulmonary arterial
hypertension, cerebro-vascular ischaemia (stroke), hypertensive
heart disease, rheumatic heart disease, cardiomyopathy, atrial
fibrillation, congenital heart disease, endocarditis, aortic
aneurysms or peripheral artery disease.
[0097] Bromodomain inhibitors may be useful in the treatment of
fibrotic conditions such as idiopathic pulmonary fibrosis,
pulmonary fibrosis, cystic fibrosis, progressive massive fibrosis,
renal fibrosis, liver fibrosis, liver cirrhosis, non-alcoholic
steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD),
post-operative stricture, keloid scar formation, scleroderma
(including morphea and systemic sclerosis), cardiac fibrosis,
atrial fibrosis, endomyocardial fibrosis, old myocardial
infarction, arthrofibrosis, Dupuytren's contracture, mediastinal,
myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis,
retroperitoneal fibrosis and adhesive capsulitis.
[0098] Bromodomain inhibitors may be useful in the treatment of
viral infections such as herpes simplex infections and
reactivations, cold sores, herpes zoster infections and
reactivations, chickenpox, shingles, human papilloma virus (HPV),
human immunodeficiency virus (HIV), cervical neoplasia, adenovirus
infections, including acute respiratory disease, poxvirus
infections such as cowpox or smallpox, or African swine fever
virus. In one embodiment the viral infection is a HPV infection of
skin or cervical epithelia. In another embodiment the viral
infection is a latent HIV infection.
[0099] Bromodomain inhibitors may be useful in the treatment of a
wide variety of bone disorders such as osteoporosis, osteopenia,
osteoarthritis and ankylosing spondylitis.
[0100] Bromodomain inhibitors may be useful in the treatment of
cancer, including hematological cancers (such as leukaemia,
lymphoma and multiple myeloma), epithelial cancers (including lung,
breast or colon carcinomas), midline carcinomas, or mesenchymal,
hepatic, renal or neurological tumours.
[0101] Bromodomain inhibitors may be useful in the treatment of one
or more cancers selected from brain cancer (gliomas),
glioblastomas, Bannayan-Zonana syndrome, Cowden disease,
Lhermitte-Duclos disease, breast cancer, inflammatory breast
cancer, colorectal cancer, Wilm's tumor, Ewing's sarcoma,
rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, head
and neck cancer, kidney cancer, lung cancer, liver cancer,
melanoma, squamous cell carcinoma, ovarian cancer, pancreatic
cancer, prostate cancer, sarcoma cancer, osteosarcoma, giant cell
tumor of bone, thyroid cancer, lymphoblastic T-cell leukemia,
chronic myelogenous leukemia, chronic lymphocytic leukemia,
hairy-cell leukemia, acute lymphoblastic leukemia, acute
myelogenous leukemia, chronic neutrophilic leukemia, acute
lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large
cell leukemia, mantle cell leukemia, multiple myeloma,
megakaryoblastic leukemia, acute megakaryocytic leukemia,
promyelocytic leukemia, mixed lineage leukaemia, erythroleukemia,
malignant lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma,
lymphoblastic T-cell lymphoma, Burkitt's lymphoma, follicular
lymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulval
cancer, cervical cancer, endometrial cancer, renal cancer,
mesothelioma, esophageal cancer, salivary gland cancer,
hepatocellular cancer, gastric cancer, nasopharangeal cancer,
buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal
tumor), NUT-midline carcinoma and testicular cancer.
[0102] In one embodiment the cancer is a leukaemia, for example a
leukaemia selected from acute monocytic leukemia, acute myelogenous
leukemia, chronic myelogenous leukemia, chronic lymphocytic
leukemia and mixed lineage leukaemia (MLL). In another embodiment
the cancer is NUT-midline carcinoma. In another embodiment the
cancer is multiple myeloma. In another embodiment the cancer is a
lung cancer such as small cell lung cancer (SCLC). In another
embodiment the cancer is a neuroblastoma. In another embodiment the
cancer is Burkitt's lymphoma. In another embodiment the cancer is
cervical cancer. In another embodiment the cancer is esophageal
cancer. In another embodiment the cancer is ovarian cancer. In
another embodiment the cancer is breast cancer. In another
embodiment the cancer is colorectal cancer. In another embodiment
the cancer is prostate cancer. In another embodiment the cancer is
castration resistant prostate cancer.
[0103] Bromodomain inhibitors may be useful in the treatment of
diseases associated with systemic inflammatory response syndrome,
such as sepsis, burns, pancreatitis, major trauma, haemorrhage and
ischaemia. In this embodiment, the bromodomain inhibitor would be
administered at the point of diagnosis to reduce the incidence of:
SIRS, the onset of shock, multi-organ dysfunction syndrome, which
includes the onset of acute lung injury, ARDS, acute renal,
hepatic, cardiac or gastro-intestinal injury and mortality. In
another embodiment the bromodomain inhibitor would be administered
prior to surgical or other procedures associated with a high risk
of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS
(multiple organ dysfunction syndrome). In a particular embodiment
the disease or condition for which a bromodomain inhibitor is
indicated is sepsis, sepsis syndrome, septic shock and
endotoxaemia. In another embodiment, the bromodomain inhibitor is
indicated for the treatment of acute or chronic pancreatitis. In
another embodiment the bromodomain is indicated for the treatment
of burns.
[0104] The present invention thus provides a compound of formula
(I) or a pharmaceutically acceptable salt thereof for use in
therapy. The compound of formula (I) or a pharmaceutically salt
thereof can be used in the treatment of diseases or conditions for
which a bromodomain inhibitor is indicated.
[0105] The present invention thus provides a compound of formula
(I) or a pharmaceutically acceptable salt thereof for use in the
treatment of a disease or condition for which a bromodomain
inhibitor is indicated. In one embodiment there is provided a
compound of formula (I) or a pharmaceutically acceptable salt
thereof for use in the treatment of acute or chronic auto-immune
and/or inflammatory conditions. In one embodiment there is provided
a compound of formula (I) or a pharmaceutically acceptable salt
thereof for use in the treatment of rheumatoid arthritis. In
another embodiment there is provided a compound of formula (I) or a
pharmaceutically acceptable salt thereof for use in the treatment
of diseases or conditions which involve inflammatory responses to
infections with bacteria, viruses, fungi, parasites or their
toxins. In another embodiment there is provided a compound of
formula (I) or a pharmaceutically acceptable salt thereof for use
in the treatment of conditions associated with
ischaemia-reperfusion injury. In another embodiment there is
provided a compound of formula (I) or a pharmaceutically acceptable
salt thereof for use in the treatment of cardiovascular diseases.
In another embodiment there is provided a compound of formula (I)
or a pharmaceutically acceptable salt thereof for use in the
treatment of fibrotic conditions. In another embodiment there is
provided a compound of formula (I) or a pharmaceutically acceptable
salt thereof for use in the treatment of viral infections. In
another embodiment there is provided a compound of formula (I) or a
pharmaceutically acceptable salt thereof for use in the treatment
of bone disorders. In another embodiment there is provided a
compound of formula (I) or a pharmaceutically acceptable salt
thereof for use in the treatment of cancer. In a further embodiment
there is provided a compound of formula (I) or a pharmaceutically
acceptable salt thereof for use in the treatment of diseases
associated with systemic inflammatory response syndrome.
[0106] Also provided is the use of a compound of formula (I) or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament for the treatment of diseases or conditions for which a
bromodomain inhibitor is indicated. In one embodiment there is
provided the use of a compound of formula (I) or a pharmaceutically
acceptable salt thereof in the manufacture of a medicament for the
treatment of acute or chronic auto-immune and/or inflammatory
conditions. In one embodiment there is provided the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof in the manufacture of a medicament for the treatment of
rheumatoid arthritis. In another embodiment there is provided the
use of a compound of formula (I) or a pharmaceutically acceptable
salt thereof in the manufacture of a medicament for the treatment
of diseases or conditions which involve inflammatory responses to
infections with bacteria, viruses, fungi, parasites or their
toxins. In another embodiment there is provided the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof in the manufacture of a medicament for the treatment of
conditions associated with ischaemia-reperfusion injury. In another
embodiment there is provided the use of a compound of formula (I)
or a pharmaceutically acceptable salt thereof in the manufacture of
a medicament for the treatment of cardiovascular diseases. In
another embodiment there is provided the use of a compound of
formula (I) or a pharmaceutically acceptable salt thereof in the
manufacture of a medicament for the treatment of fibrotic
conditions. In another embodiment there is provided the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof in the manufacture of a medicament for the treatment of
viral infections. In another embodiment there is provided the use
of a compound of formula (I) or a pharmaceutically acceptable salt
thereof in the manufacture of a medicament for the treatment of
cancer. In a further embodiment there is provided the use of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof in the manufacture of a medicament for the treatment of
diseases associated with systemic inflammatory response
syndrome.
[0107] Also provided is a method of treating diseases or conditions
for which a bromodomain inhibitor is indicated in a subject in need
thereof which comprises administering a therapeutically effective
amount of compound of formula (I) or a pharmaceutically acceptable
salt thereof. In one embodiment there is provided a method of
treating acute or chronic auto-immune and/or inflammatory
conditions in a subject in need thereof which comprises
administering a therapeutically effective amount of a compound of
formula (I) or a pharmaceutically acceptable salt thereof. In one
embodiment there is provided a method of treating rheumatoid
arthritis in a subject in need thereof which comprises
administering a therapeutically effective amount of a compound of
formula (I) or a pharmaceutically acceptable salt thereof. In
another embodiment there is provided a method of treating diseases
or conditions which involve inflammatory responses to infections
with bacteria, viruses, fungi, parasites or their toxins in a
subject in need thereof which comprises administering a
therapeutically effective amount of a compound of formula (I) or a
pharmaceutically acceptable salt thereof. In another embodiment
there is provided a method of treating conditions associated with
ischaemia-reperfusion injury in a subject in need thereof which
comprises administering a therapeutically effective amount of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof. In another embodiment there is provided a method of
treating cardiovascular diseases in a subject in need thereof which
comprises administering a therapeutically effective amount of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof. In another embodiment there is provided a method of
treating fibrotic conditions in a subject in need thereof which
comprises administering a therapeutically effective amount of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof. In another embodiment there is provided a method of
treating viral infections in a subject in need thereof which
comprises administering a therapeutically effective amount of a
compound of formula (I) or a pharmaceutically acceptable salt
thereof. In another embodiment there is provided a method of
treating cancer in a subject in need thereof which comprises
administering a therapeutically effective amount of a compound of
formula (I) or a pharmaceutically acceptable salt thereof. In a
further embodiment there is provided a method of treating diseases
associated with systemic inflammatory response syndrome in a
subject in need thereof which comprises administering a
therapeutically effective amount of a compound of formula (I) or a
pharmaceutically acceptable salt thereof.
[0108] Suitably the subject in need thereof is a mammal,
particularly a human.
[0109] The invention further provides for a method for inhibiting a
bromodomain containing protein which comprises contacting the
bromodomain containing protein with a compound of formula (I) or a
pharmaceutically acceptable salt thereof.
[0110] As used herein the reference to the "treatment" of a
particular disease or condition includes the prevention or
prophylaxis of such a disease or condition.
PHARMACEUTICAL COMPOSITIONS/ROUTES OF ADMINISTRATION/DOSAGES
Compositions
[0111] While it is possible that for use in therapy, a compound of
formula (I) as well as pharmaceutically acceptable salts thereof
may be administered as the raw chemical, it is common to present
the active ingredient as a pharmaceutical composition. The
compounds of formula (I) and pharmaceutically acceptable salts
thereof will normally, but not necessarily, be formulated into
pharmaceutical compositions prior to administration to a patient.
Accordingly, in another aspect there is provided a pharmaceutical
composition comprising a compound of formula (I), or a
pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable excipients. The compounds of formula
(I) and pharmaceutically acceptable salts are as described above.
The excipient(s) must be acceptable in the sense of being
compatible with the other ingredients of the composition and not
deleterious to the recipient thereof. In accordance with another
aspect of the invention there is also provided a process for the
preparation of a pharmaceutical composition including admixing a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, with one or more pharmaceutically acceptable excipients. A
pharmaceutical composition comprising a compound of formula (I) or
a pharmaceutically acceptable salt thereof may be prepared by, for
example, admixture at ambient temperature and atmospheric pressure.
The pharmaceutical composition can be used in the treatment of any
of the conditions described herein.
[0112] In a further aspect the invention is directed to
pharmaceutical compositions for the treatment of a disease or
condition for which a bromodomain inhibitor is indicated comprising
a compound of formula (I) or a pharmaceutically acceptable salt
thereof.
[0113] Since the compounds of formula (I) are intended for use in
pharmaceutical compositions it will be readily understood that they
are each preferably provided in substantially pure form, for
example, at least 85% pure, especially at least 98% pure (% in a
weight for weight basis).
[0114] Pharmaceutical compositions may be presented in unit dose
forms containing a predetermined amount of active ingredient per
unit dose. Preferred unit dosage compositions are those containing
a daily dose or sub-dose, or an appropriate fraction thereof, of an
active ingredient. Such unit doses may therefore be administered
more than once a day. Preferred unit dosage compositions are those
containing a daily dose or sub-dose (for administration more than
once a day), as herein above recited, or an appropriate fraction
thereof, of an active ingredient.
[0115] Pharmaceutical compositions may be adapted for
administration by any appropriate route, for example by the oral
(including buccal or sublingual), rectal, inhaled, intranasal,
topical (including buccal, sublingual or transdermal), ocular
(including topical, intraocular, subconjunctival, episcleral,
sub-Tenon), vaginal or parenteral (including subcutaneous,
intramuscular, intravenous or intradermal) route. Such compositions
may be prepared by any method known in the art of pharmacy, for
example by bringing into association the active ingredient with the
carrier(s) or excipient(s).
[0116] The pharmaceutical compositions of the invention may be
prepared and packaged in bulk form wherein a safe and effective
amount of a compound of formula (I) or a pharmaceutically
acceptable salt thereof can be extracted and then given to the
patient such as with powders or syrups. Alternatively, the
pharmaceutical compositions of the invention may be prepared and
packaged in unit dosage form wherein each physically discrete unit
contains a compound of formula (I) or a pharmaceutically acceptable
salt thereof. When prepared in unit dosage form, the pharmaceutical
compositions of the invention typically may contain, for example,
from 0.25 mg to 1 g, or from 0.5 mg to 500 mg, or from 1 mg to 100
mg, of a compound of formula (I) or a pharmaceutically acceptable
salt thereof.
[0117] The compound of formula (I) or a pharmaceutically acceptable
salt thereof and the pharmaceutically acceptable excipient or
excipients will typically be formulated into a dosage form adapted
for administration to the patient by the desired route of
administration. For example, dosage forms include those adapted for
(1) oral administration such as tablets, capsules, caplets, pills,
troches, powders, syrups, elixers, suspensions, solutions,
emulsions, sachets, and cachets; (2) parenteral administration such
as sterile solutions, suspensions, and powders for reconstitution;
(3) transdermal administration such as transdermal patches; (4)
rectal administration such as suppositories; (5) inhalation such as
aerosols, solutions, and dry powders; and (6) topical
administration such as creams, ointments, lotions, solutions,
pastes, sprays, foams, and gels.
[0118] Suitable pharmaceutically acceptable excipients will vary
depending upon the particular dosage form chosen. In addition,
suitable pharmaceutically acceptable excipients may be chosen for a
particular function that they may serve in the composition. For
example, certain pharmaceutically acceptable excipients may be
chosen for their ability to facilitate the production of uniform
dosage forms. Certain pharmaceutically acceptable excipients may be
chosen for their ability to facilitate the production of stable
dosage forms. Certain pharmaceutically acceptable excipients may be
chosen for their ability to facilitate the carrying or transporting
of the compound or compounds of formula (I) or pharmaceutically
acceptable salts thereof once administered to the subject from one
organ, or portion of the body, to another organ, or portion of the
body. Certain pharmaceutically acceptable excipients may be chosen
for their ability to enhance subject compliance.
[0119] Suitable pharmaceutically-acceptable excipients include the
following types of excipients: carriers, diluents, fillers,
binders, disintegrants, lubricants, glidants, granulating agents,
coating agents, wetting agents, solvents, co-solvents, suspending
agents, emulsifiers, sweetners, flavouring agents, flavour-masking
agents, colouring agents, anti-caking agents, humectants, chelating
agents, plasticisers, viscosity increasing agents, antioxidants,
preservatives, stabilisers, surfactants, and buffering agents. The
skilled artisan will appreciate that certain
pharmaceutically-acceptable excipients may serve more than one
function and may serve alternative functions depending on how much
of the excipient is present in the formulation and what other
excipients are present in the formulation.
[0120] Skilled artisans possess the knowledge and skill in the art
to enable them to select suitable pharmaceutically-acceptable
excipients in appropriate amounts for use in the invention. In
addition, there are a number of resources that are available to the
skilled artisan which describe pharmaceutically-acceptable
excipients and may be useful in selecting suitable
pharmaceutically-acceptable excipients. Examples include
Remington's Pharmaceutical Sciences (Mack Publishing Company), The
Handbook of Pharmaceutical Additives (Gower Publishing Limited),
and The Handbook of Pharmaceutical Excipients (the American
Pharmaceutical Association and the Pharmaceutical Press).
[0121] The pharmaceutical compositions of the invention are
prepared using techniques and methods known to those skilled in the
art. Some of the methods commonly used in the art are described in
Remington's Pharmaceutical Sciences (Mack Publishing Company).
[0122] In one embodiment the pharmaceutical composition is adapted
for parenteral administration, particularly intravenous
administration.
[0123] In one embodiment the pharmaceutical composition is adapted
for oral administration.
[0124] In one embodiment the pharmaceutical composition is adapted
for topical administration.
[0125] Pharmaceutical compositions adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solutions (which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the composition isotonic with the blood of
the intended recipient) and aqueous and non-aqueous sterile
suspensions (which may include suspending agents and thickening
agents). The compositions may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0126] Pharmaceutical compositions adapted for oral administration
may be presented as discrete units such as capsules or tablets;
powders or granules; solutions or suspensions in aqueous or
non-aqueous liquids; edible foams or whips; or oil-in-water liquid
emulsions or water-in-oil liquid emulsions.
[0127] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Powders suitable for
incorporating into tablets or capsules may be prepared by reducing
the compound to a suitable fine size (e.g. by micronisation) and
mixing with a similarly prepared pharmaceutical carrier such as an
edible carbohydrate, for example, starch or mannitol. Flavoring,
preservative, dispersing and coloring agent can also be
present.
[0128] Capsules may be made by preparing a powder mixture, as
described above, and filling formed gelatin sheaths. Glidants and
lubricants such as colloidal silica, talc, magnesium stearate,
calcium stearate or solid polyethylene glycol can be added to the
powder mixture before the filling operation. A disintegrating or
solubilizing agent such as agar-agar, calcium carbonate or sodium
carbonate can also be added to improve the availability of the
medicament when the capsule is ingested.
[0129] Moreover, when desired or necessary, suitable binders,
glidants, lubricants, sweetening agents, flavours, disintegrating
agents (disintegrants) and coloring agents can also be incorporated
into the mixture. Suitable binders include starch, gelatin, natural
sugars such as glucose or beta-lactose, corn sweeteners, natural
and synthetic gums such as acacia, tragacanth or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include sodium oleate, sodium
stearate, magnesium stearate, sodium benzoate, sodium acetate,
sodium chloride and the like. Disintegrants include starch, methyl
cellulose, agar, bentonite, xanthan gum and the like. Tablets are
formulated, for example, by preparing a powder mixture, granulating
or slugging, adding a lubricant and disintegrant and pressing into
tablets. A powder mixture is prepared by mixing the compound,
suitably comminuted, with a diluent or base as described above, and
optionally, with a binder such as carboxymethylcellulose, an
aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant
such as paraffin, a resorption accelerator such as a quaternary
salt and/or an absorption agent such as bentonite, kaolin or
dicalcium phosphate. The powder mixture can be granulated by
wetting with a binder such as syrup, starch paste, acadia mucilage
or solutions of cellulosic or polymeric materials and forcing
through a screen. As an alternative to granulating, the powder
mixture can be run through the tablet machine and the result is
imperfectly formed slugs broken into granules. The granules can be
lubricated to prevent sticking to the tablet forming dies by means
of the addition of stearic acid, a stearate salt, talc or mineral
oil. The lubricated mixture is then compressed into tablets. The
compounds of formula (I) and pharmaceutically acceptable salts
thereof can also be combined with a free flowing inert carrier and
compressed into tablets directly without going through the
granulating or slugging steps. A clear or opaque protective coating
consisting of a sealing coat of shellac, a coating of sugar or
polymeric material and a polish coating of wax can be provided.
Dyestuffs can be added to these coatings to distinguish different
unit dosages.
[0130] Oral fluids such as solution, syrups and elixirs can be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups can be prepared by
dissolving the compound in a suitably flavored aqueous solution,
while elixirs are prepared through the use of a non-toxic alcoholic
vehicle. Suspensions can be formulated by dispersing the compound
in a non-toxic vehicle. Solubilizers and emulsifiers such as
ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol
ethers, preservatives, flavor additive such as peppermint oil or
natural sweeteners or saccharin or other artificial sweeteners, and
the like can also be added.
[0131] Compositions for oral administration may be designed to
provide a modified release profile so as to sustain or otherwise
control the release of the therapeutically active agent.
[0132] Where appropriate, dosage unit compositions for oral
administration can be microencapsulated. The composition may be
prepared to prolong or sustain the release as for example by
coating or embedding particulate material in polymers, wax or the
like.
[0133] For compositions suitable and/or adapted for oral
administration, the compound of formula (I) or a pharmaceutically
acceptable salt thereof, may be in a particle-size-reduced form
e.g. obtained by micronisation. The preferable particle size of the
size-reduced (e.g. micronised) compound or salt is defined by a
D.sub.50 value of about 0.5 to about 10 microns (for example as
measured using laser diffraction).
[0134] The compounds of formula (I) and pharmaceutically acceptable
salts thereof, can also be administered in the form of liposome
delivery systems, such as small unilamellar vesicles, large
unilamellar vesicles and multilamellar vesicles. Liposomes can be
formed from a variety of phospholipids, such as cholesterol,
stearylamine or phosphatidylcholines.
[0135] Pharmaceutical compositions adapted for topical
administration may be formulated as ointments, creams, suspensions,
emulsions, lotions, powders, solutions, pastes, gels, foams,
sprays, aerosols or oils. Such pharmaceutical compositions may
include conventional additives which include, but are not limited
to, preservatives, solvents to assist drug penetration,
co-solvents, emollients, propellants, viscosity modifying agents
(gelling agents), surfactants and carriers. In one embodiment there
is provided a pharmaceutical composition adapted for topical
administration which comprises between 0.01-10%, or between 0.01-1%
of the compound of formula (I), or a pharmaceutically acceptable
salt thereof, by weight of the composition.
[0136] For treatments of the eye or other external tissues, for
example mouth and skin, the compositions are preferably applied as
a topical ointment, cream, gel, spray or foam. When formulated in
an ointment, the active ingredient may be employed with either a
paraffinic or a water-miscible ointment base. Alternatively, the
active ingredient may be formulated in a cream with an oil-in-water
cream base or a water-in-oil base.
[0137] Pharmaceutical compositions adapted for topical
administrations to the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent. Compositions to be administered to
the eye will have ophthalmically compatible pH and osmolality. One
or more ophthalmically acceptable pH adjusting agents and/or
buffering agents can be included in a composition of the invention,
including acids such as acetic, boric, citric, lactic, phosphoric
and hydrochloric acids; bases such as sodium hydroxide, sodium
phosphate, sodium borate, sodium citrate, sodium acetate, and
sodium lactate; and buffers such as citrate/dextrose, sodium
bicarbonate and ammonium chloride. Such acids, bases, and buffers
can be included in an amount required to maintain pH of the
composition in an ophthalmically acceptable range. One or more
ophthalmically acceptable salts can be included in the composition
in an amount sufficient to bring osmolality of the composition into
an ophthalmically acceptable range. Such salts include those having
sodium, potassium or ammonium cations and chloride, citrate,
ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or
bisulflte anions.
[0138] The ocular delivery device may be designed for the
controlled release of one or more therapeutic agents with multiple
defined release rates and sustained dose kinetics and permeability.
Controlled release may be obtained through the design of polymeric
matrices incorporating different choices and properties of
biodegradable/bioerodable polymers (e.g. poly(ethylene vinyl)
acetate (EVA), superhydrolyzed PVA), hydroxyalkyl cellulose (HPC),
methylcellulose (MC), hydroxypropyl methyl cellulose (HPMC),
polycaprolactone, poly(glycolic) acid, poly(lactic) acid,
polyanhydride, of polymer molecular weights, polymer crystallinity,
copolymer ratios, processing conditions, surface finish, geometry,
excipient addition and polymeric coatings that will enhance drug
diffusion, erosion, dissolution and osmosis.
[0139] Pharmaceutical compositions for ocular delivery also include
in situ gellable aqueous composition. Such a composition comprises
a gelling agent in a concentration effective to promote gelling
upon contact with the eye or with lacrimal fluid. Suitable gelling
agents include but are not limited to thermosetting polymers. The
term "in situ gellable" as used herein is includes not only liquids
of low viscosity that form gels upon contact with the eye or with
lacrimal fluid, but also includes more viscous liquids such as
semi-fluid and thixotropic gels that exhibit substantially
increased viscosity or gel stiffness upon administration to the
eye. See, for example, Ludwig (2005) Adv. Drug Deliv. Rev. 3;
57:1595-639, herein incorporated by reference for purposes of its
teachings of examples of polymers for use in ocular drug
delivery.
[0140] Dosage forms for nasal or inhaled administration may
conveniently be formulated as aerosols, solutions, suspensions,
gels or dry powders.
[0141] For compositions suitable and/or adapted for inhaled
administration, it is preferred that the compound of formula (I) or
a pharmaceutically acceptable salt thereof, is in a
particle-size-reduced form e.g. obtained by micronisation. The
preferable particle size of the size-reduced (e.g. micronised)
compound or salt is defined by a D.sub.50 value of about 0.5 to
about 10 microns (for example as measured using laser
diffraction).
[0142] Aerosol formulations, e.g. for inhaled administration, can
comprise a solution or fine suspension of the active substance in a
pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol
formulations can be presented in single or multidose quantities in
sterile form in a sealed container, which can take the form of a
cartridge or refill for use with an atomising device or inhaler.
Alternatively the sealed container may be a unitary dispensing
device such as a single dose nasal inhaler or an aerosol dispenser
fitted with a metering valve (metered dose inhaler) which is
intended for disposal once the contents of the container have been
exhausted.
[0143] Where the dosage form comprises an aerosol dispenser, it
preferably contains a suitable propellant under pressure such as
compressed air, carbon dioxide or an organic propellant such as a
hydrofluorocarbon (HFC). Suitable HFC propellants include
1,1,1,2,3,3,3-heptafluoropropane and 1,1,1,2-tetrafluoroethane. The
aerosol dosage forms can also take the form of a pump-atomiser. The
pressurised aerosol may contain a solution or a suspension of the
active compound. This may require the incorporation of additional
excipients e.g. co-solvents and/or surfactants to improve the
dispersion characteristics and homogeneity of suspension
formulations. Solution formulations may also require the addition
of co-solvents such as ethanol.
[0144] For pharmaceutical compositions suitable and/or adapted for
inhaled administration, the pharmaceutical composition may be a dry
powder inhalable composition. Such a composition can comprise a
powder base such as lactose, glucose, trehalose, mannitol or
starch, the compound of formula (I) or a pharmaceutically
acceptable salt thereof (preferably in particle-size-reduced form,
e.g. in micronised form), and optionally a performance modifier
such as L-leucine or another amino acid and/or metal salt of
stearic acid such as magnesium or calcium stearate. Preferably, the
dry powder inhalable composition comprises a dry powder blend of
lactose e.g. lactose monohydrate and the compound of formula (I) or
salt thereof. Such compositions can be administered to the patient
using a suitable device such as the DISKUS.RTM. device, marketed by
GlaxoSmithKline which is for example described in GB 2242134 A.
[0145] The compounds of formula (I) and pharmaceutically acceptable
salts thereof may be formulated as a fluid formulation for delivery
from a fluid dispenser, for example a fluid dispenser having a
dispensing nozzle or dispensing orifice through which a metered
dose of the fluid formulation is dispensed upon the application of
a user-applied force to a pump mechanism of the fluid dispenser.
Such fluid dispensers are generally provided with a reservoir of
multiple metered doses of the fluid formulation, the doses being
dispensable upon sequential pump actuations. The dispensing nozzle
or orifice may be configured for insertion into the nostrils of the
user for spray dispensing of the fluid formulation into the nasal
cavity. A fluid dispenser of the aforementioned type is described
and illustrated in International Patent Application Publication No.
WO 2005/044354 A1.
[0146] A therapeutically effective amount of a compound of formula
(I) or a pharmaceutically acceptable salt thereof, will depend upon
a number of factors including, for example, the age and weight of
the patient, the precise condition requiring treatment and its
severity, the nature of the formulation, and the route of
administration, and will ultimately be at the discretion of the
attendant physician or veterinarian. In the pharmaceutical
composition, each dosage unit for oral or parenteral administration
preferably contains from 0.01 mg to 3000 mg, more preferably 0.5 mg
to 1000 mg, of a compound of formula (I) or a pharmaceutically
acceptable salt thereof, calculated as the free base. Each dosage
unit for nasal or inhaled administration preferably contains from
0.001 mg to 50 mg, more preferably 0.01 mg to 5 mg, of a compound
of the formula (I) or a pharmaceutically acceptable salt thereof,
calculated as the free base.
[0147] The pharmaceutically acceptable compounds of formula (I) and
pharmaceutically acceptable salts thereof, can be administered in a
daily dose (for an adult patient) of, for example, an oral or
parenteral dose of 0.01 mg to 3000 mg per day, 0.5 mg to 1000 mg
per day or 100 mg to 2500 mg per day, or a nasal or inhaled dose of
0.001 mg to 50 mg per day or 0.01 mg to 5 mg per day, of the
compound of the formula (I) or a pharmaceutically acceptable salt
thereof, calculated as the free base. This amount may be given in a
single dose per day or more usually in a number (such as two,
three, four, five or six) of sub-doses per day such that the total
daily dose is the same. An effective amount of a salt thereof, may
be determined as a proportion of the effective amount of the
compound of formula (I) per se.
[0148] The compounds of formula (I) and pharmaceutically acceptable
salts thereof may be employed alone or in combination with other
therapeutic agents. Combination therapies according to the present
invention thus comprise the administration of at least one compound
of formula (I) or a pharmaceutically acceptable salt thereof, and
the use of at least one other theraputically active agent. The
compound(s) of formula (I) and pharmaceutically acceptable salts
thereof, and the other therapeutically active agent(s) may be
administered together in a single pharmaceutical composition or
separately and, when administered separately this may occur
simultaneously or sequentially in any order. The amounts of the
compound(s) of formula (I) and pharmaceutically acceptable salts
thereof, and the other therapeutically active agent(s) and the
relative timings of administration will be selected in order to
achieve the desired combined therapeutic effect. Thus in a further
aspect, there is provided a combination comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof, together
with one or more other therapeutically active agents.
[0149] Thus in one aspect, the compound of formula (I) or a
pharmaceutically acceptable salt thereof, and pharmaceutical
compositions comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof, according to the
invention may be used in combination with or include one or more
other therapeutic agents, for example selected from antibiotics,
anti-virals, glucocorticosteroids, muscarinic antagonists, beta-2
agonists and Vitamin D3 analogues. In a further embodiment a
compound of formula (I) or a pharmaceutically acceptable salt
thereof may be used in combination with a further therapeutic agent
which is suitable for the treatment of cancer. Examples of such
further therapeutic agents are described in Cancer Principles and
Practice of Oncology by V. T. Devita and S. Hellman (editors),
6.sup.th edition (2001), Lippincott Williams & Wilkins
Publishers. A person of ordinary skill in the art would be able to
discern which combinations of agents would be useful based on the
particular characteristics of the drugs and the cancer involved.
Further therapeutic agents to be used in combination with the
compound of formula (I) or a pharmaceutically acceptable salt
thereof include, but are not limited to, anti-microtubule agents
(such as diterpenoids and vinca alkaloids); platinum coordination
complexes; alkylating agents (such as nitrogen mustards,
oxazaphosphorines, alkylsulphonates, nitrosoureas, and triazenes);
antibiotic agents (such as anthracyclins, actinomycins and
bleomycins); topoisomerase II inhibitors (such as
epipodophyllotoxins); antimetabolites (such as purine and
pyrimidine analogues and anti-folate compounds); topoisomerase I
inhibitors (such as camptothecins; hormones and hormonal
analogues); signal transduction pathway inhibitors (such as
tyropsine receptor inhibitors); non-receptor tyrosine kinase
angiogenesis inhibitors; immunotherapeutic agents (such as PD-1
inhibitors, including nivolumab and pembrolizumab, and CTLA-4
inhibitors, including ipilimumab); proapoptotic agents; epigenetic
or transcriptional modulators (such as histone deacetylase
inhibitors) and cell cycle signaling inhibitors.
[0150] It will be appreciated that when the compound of formula (I)
or a pharmaceutically acceptable salt thereof, is administered in
combination with other therapeutic agents normally administered by
the inhaled, intravenous, oral or intranasal route, that the
resultant pharmaceutical composition may be administered by the
same routes. Alternatively the individual components of the
composition may be administered by different routes.
[0151] It will be clear to a person skilled in the art that, where
appropriate, the other therapeutic agent(s) may be used in the form
of salts, for example as alkali metal or amine salts or as acid
addition salts, or prodrugs, or as esters, for example lower alkyl
esters, or as solvates, for example hydrates, to optimise the
activity and/or stability and/or physical characteristics, such as
solubility, of the therapeutic agent. It will be clear also that,
where appropriate, the therapeutic agents may be used in optically
pure form.
[0152] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical composition and
thus pharmaceutical compositions comprising a combination as
defined above together with a pharmaceutically acceptable excipient
represent a further aspect of the invention.
General Synthetic Routes
[0153] The compounds of the invention may be made by a variety of
methods. Any previously defined variable will continue to have the
previously defined meaning unless otherwise indicated. Illustrative
general synthetic methods are set out in the following schemes, and
can be readily adapted to prepare other compounds of the invention.
Specific compounds of the invention are prepared in the Examples
section.
[0154] Compounds of formula (I) may be prepared as described in any
of the Schemes below:
##STR00004##
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as described
above, Hal is chlorine or bromine and X is either H or joined
together to form a cyclic boronate ester, such as
--C(Me).sub.2C(Me).sub.2-.
[0155] In respect of the steps shown in Scheme 1 above the
following reaction conditions may be utilised: [0156] Step 1: is a
Negishi cross coupling and may be carried out using a benzylzinc
halide of formula R.sup.4CH(R.sup.3)ZnHal, in the presence of a
palladium catalyst, such as PdCl.sub.2(PPh.sub.3).sub.2, optionally
in the presence of an alternative phosphine ligand, in a suitable
solvent, such as THF, at a suitable temperature, such as 70.degree.
C. [0157] Step 2: is an acid-mediated ester cleavage and may be
carried out using any suitable acid, such as TFA, optionally in a
suitable solvent, such as DCM, at a suitable temperature, such as
room temperature. [0158] Step 3: is an amide coupling reaction and
may be carried out using an amine reagent, R.sup.2--NH.sub.2, in
the presence of a suitable tertiary amine, such as triethylamine or
DIPEA, in the presence of a suitable amide coupling reactant, such
as HATU, in a suitable solvent, such as DCM or DMF, at a suitable
temperature, such as room temperature. [0159] Step 4: is an
optional deprotection step to remove a protecting group, such as
BOC and may be carried out using an acid such as TFA or HCl, in the
presence of a suitable solvent, such as DCM or 1,4-dioxane, at a
suitable temperature, such as room temperature. [0160] Step 5: is
an optional chiral separation, using a suitable chiral HPLC column
and a suitable solvent system. [0161] Step 6: is a carbonylation
reaction and may be carried out using an alcohol reagent, such as
EtOH, in the presence of a tertiary amine, such as triethylamine,
in the presence of a palladium catalyst, such as
[(R)-(+)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]palladium(II-
) chloride, optionally in the presence of an alternative phosphine
ligand, in the presence of carbon monoxide, in a suitable solvent,
such as DMF, at a suitable temperature, such as 70.degree. C.
[0162] Step 7: is a reduction and may be carried out using a
reducing agent or combination of reagents, such as sodium
borohydride and calcium chloride, in a suitable solvent or solvent
mixture, such as ethanol and 2-MeTHF, at a suitable temperature,
such as 0.degree. C. to room temperature. [0163] Step 8: is a
chlorination reaction and may be carried out using a chlorinating
reagent, such as thionyl chloride, in the presence of a suitable
solvent, such as DCM, at a suitable temperature, such as room
temperature. [0164] Step 9: is a cross-coupling reaction, such as a
Suzuki coupling and may be carried out using an arylmetal species,
such as a arylboronic acid or arylboronate ester,
R.sup.4--B(OX).sub.2 in the presence of a suitable palladium
catalyst, such as PdCl.sub.2(PPh.sub.3).sub.2, optionally in the
presence of an alternative phosphine ligand, in the presence of a
suitable base, such as potassium carbonate, in the presence of a
suitable solvent or solvent mixture, such as 1,4-dioxane and water,
at a suitable temperature, such as 120.degree. C. [0165] Step 10:
is an oxidation and may be carried out using a suitable oxidant,
such as Dess-Martin periodinane in a suitable solvent, such as DCM,
at a suitable temperature, such as room temperature. [0166] Step
11: is a Grignard addition to an aldehyde, using a suitable
Grignard reagent, such as phenylmagnesium bromide, in a suitable
solvent, such as THF, at a suitable temperature, such as 0.degree.
C. [0167] Step 12: is a substitution reaction of an alcohol with a
halide, such as chloride, using a suitable chlorinating reagent,
such as thionyl chloride, in a suitable solvent, such as DCM, at a
suitable temperature, such as 0.degree. C. [0168] Step 13: is a
substitution reaction of a leaving group, such as chloride with a
nucleophile, such as a methoxy group, using a suitable nucleophilic
reagent, such as methanol, optionally in the presence of a suitable
solvent, at a suitable temperature, such as room temperature.
[0169] It will be appreciated by those skilled in the art that it
may be advantageous to protect one or more functional groups of the
compounds described above. Examples of protecting groups and the
means for their removal can be found in T. W. Greene `Protective
Groups in Organic Synthesis` (4th edition, J. Wiley and Sons,
2006), incorporated herein by reference as it relates to such
procedures.
[0170] Suitable amine protecting groups include acyl (e.g. acetyl,
carbamate (e.g. 2',2',2'-trichloroethoxycarbonyl, benzyloxycarbonyl
or t-butoxycarbonyl) and arylalkyl (e.g. benzyl), which may be
removed by acid mediated cleavage (e.g. using an acid such as
hydrochloric acid in 1,4-dioxane or trifluoroacetic acid in
dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl or
benzyloxycarbonyl group or reductive removal of a
2',2',2'-trichloroethoxycarbonyl group using zinc in acetic acid)
as appropriate. Other suitable amine protecting groups include
trifluoroacetyl (--C(O)CF.sub.3) which may be removed by base
catalysed hydrolysis.
[0171] It will be appreciated that in any of the routes described
above, the precise order of the synthetic steps by which the
various groups and moieties are introduced into the molecule may be
varied. It will be within the skill of the practitioner in the art
to ensure that groups or moieties introduced at one stage of the
process will not be affected by subsequent transformations and
reactions, and to select the order of synthetic steps
accordingly.
[0172] Certain intermediate compounds described above form a yet
further aspect of the invention.
[0173] For any of the hereinbefore described reactions or
processes, conventional methods of heating and cooling may be
employed, for example temperature-regulated oil-baths or
temperature-regulated hot-blocks, and ice/salt baths or dry
ice/acetone baths respectively. Conventional methods of isolation,
for example extraction from or into aqueous or non-aqueous solvents
may be used. Conventional methods of drying organic solvents,
solutions, or extracts, such as shaking with anhydrous magnesium
sulfate, or anhydrous sodium sulfate, or passing through a
hydrophobic frit, may be employed. Conventional methods of
purification, for example crystallisation and chromatography, for
example silica chromatography or reverse-phase chromatography, may
be used as required. Crystallisation may be performed using
conventional solvents such as ethyl acetate, methanol, ethanol, or
butanol, or aqueous mixtures thereof. It will be appreciated that
specific reaction times and temperatures may typically be
determined by reaction-monitoring techniques, for example
thin-layer chromatography and LC-MS.
General Experimental Details
[0174] All temperatures referred to are in .degree. C.
[0175] As used herein the symbols and conventions used in these
processes, schemes and examples are consistent with those used in
the contemporary scientific literature, for example, the Journal of
the American Chemical Society. Unless otherwise noted, all starting
materials were obtained from commercial suppliers and used without
further purification. Specifically, the following abbreviations may
be used in the examples and throughout the specification:
Abbreviations
[0176] ACD Advanced Chemistry Development, Inc. [0177] AMU atomic
mass unit [0178] BOC/Boc tert-butyloxycarbonyl [0179] cart
cartridge [0180] cat catalyst [0181] CSH Water's Charged Surface
Hybrid Technology [0182] CV column volume [0183] DCM
dichloromethane [0184] DIPEA diisopropylethylamine [0185] DMAP
4-dimethylaminopyridine [0186] DMF dimethylformamide [0187] DMSO
dimethylsulfoxide [0188] DMSO-d.sub.6 deuterated dimethylsulfoxide
[0189] dppf 1,1'-bis(diphenylphosphino)ferrocene [0190] h hour(s)
[0191] HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0192] IPA isopropyl alcohol [0193] Isolera
Biotage Flash purification system [0194] LC liquid chromatography
[0195] LCMS liquid chromatography-mass spectrometry [0196] M molar
(concentration) [0197] MDAP mass directed autopreparative
chromatography [0198] 2-MeTHF 2-methyl tetrahydrofuran [0199] min
minute(s) [0200] MS mass spectrometry [0201] Ms-Cl methanesulfonyl
chloride [0202] MTBE methyl tert-butyl ether [0203] N normal
(concentration) [0204] NMR nuclear magnetic resonance [0205] NUT
nuclear protein in testis [0206] obs obscured [0207] RBF round
bottomed flask [0208] Rt retention time [0209] rt room temperature
[0210] sat saturated [0211] SCX Isolute strong cation exchange
sorbent SPE [0212] sec second [0213] SiO.sub.2 silicon dioxide
[0214] SNAP Biotage (silica) flash chromatography cartridge [0215]
SP4 Biotage Flash purification system [0216] SPE solid phase
extraction [0217] TFA trifluoroacetic acid [0218] THF
tetrahydrofuran [0219] TLC thin layer chromatography [0220] T3P
propylphosphonic anhydride [0221] UPLC ultra performance liquid
chromatograpy [0222] UV ultra-violet [0223] wt weight
[0224] The names of the following compounds have been obtained
using the compound naming programme "ACD Name Pro 6.02" or using
the naming functionality of ChemDraw Ultra 12.0.
LCMS Methodology
[0225] Formic Method
[0226] LC Conditions
[0227] The UPLC analysis was conducted on an Acquity UPLC CSH C18
column (50 mm.times.2.1 mm, i.d. 1.7 .mu.m packing diameter) at
40.degree. C.
[0228] The solvents employed were:
[0229] A=0.1% v/v solution of formic acid in water
[0230] B=0.1% v/v solution of formic acid in acetonitrile
[0231] The gradient employed was:
TABLE-US-00001 Time Flow rate (min) (mL/min) % A % B 0 1 97 3 1.5 1
5 95 1.9 1 5 95 2.0 1 97 3
[0232] The UV detection was a summed signal from wavelength of 210
nm to 350 nm.
[0233] MS Conditions
[0234] MS: Waters ZQ
[0235] Ionisation mode: Alternate-scan positive and negative
electrospray
[0236] Scan range: 100 to 1000 AMU
[0237] Scan time 0.27 sec
[0238] Inter scan delay 0.10 sec
[0239] High pH Method
[0240] LC Conditions
[0241] The UPLC analysis was conducted on an Acquity UPLC CSH C18
column (50 mm.times.2.1 mm, i.d. 1.7 .mu.m packing diameter) at
40.degree. C.
[0242] The solvents employed were:
[0243] A=10 mM ammonium hydrogen carbonate in water adjusted to
pH10 with ammonia solution
[0244] B=acetonitrile
[0245] The gradient employed was:
TABLE-US-00002 Time Flow rate (min) (mL/min) % A % B 0 1 97 3 0.05
1 97 3 1.5 1 5 95 1.9 1 5 95 2.0 1 97 3
[0246] The UV detection was a summed signal from wavelength of 210
nm to 350 nm.
[0247] MS Conditions
[0248] MS: Waters ZQ
[0249] Ionisation mode: Alternate-scan positive and negative
electrospray
[0250] Scan range: 100 to 1000 AMU
[0251] Scan time: 0.27 sec
[0252] Inter scan delay: 0.10 sec
[0253] TFA Method
[0254] LC Conditions
[0255] The UPLC analysis was conducted on an Acquity UPLC CSH C18
column (50 mm.times.2.1 mm, i.d. 1.7 .mu.m packing diameter) at
40.degree. C.
[0256] The solvents employed were:
[0257] A=0.1% v/v solution of trifluoroacetic acid in water
[0258] B=0.1% v/v solution of trifluoroacetic acid in
acetonitrile
[0259] The gradient employed was:
TABLE-US-00003 Time Flow rate (min) (mL/min) % A % B 0 1 95 5 1.5 1
5 95 1.9 1 5 95 2.0 1 95 5
[0260] The UV detection was a summed signal from wavelength of 210
nm to 350 nm.
[0261] MS Conditions
[0262] MS: Waters ZQ
[0263] Ionisation mode: Alternate-scan positive and negative
electrospray
[0264] Scan range: 100 to 1000 AMU
[0265] Scan time: 0.27 sec
[0266] Inter scan delay: 0.10 sec
[0267] General MDAP Purification Methods
[0268] Listed below are examples of mass-directed autopreparative
chromatography (MDAP) methods that have been used or may be used in
compound purification.
[0269] MDAP (High pH). The HPLC analysis was conducted on an
Xselect CSH C18 column (150 mm.times.30 mm i.d. 5 .mu.m packing
diameter) at ambient temperature, eluting with 10 mM ammonium
bicarbonate in water adjusted to pH 10 with ammonia solution
(Solvent A) and acetonitrile (Solvent B) using an elution gradient
of between 0 and 100% Solvent B over 15 or 25 min.
[0270] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm. The mass spectra were recorded on a Waters ZQ
Mass Spectrometer using alternate-scan positive and negative
electrospray. Ionisation data was rounded to the nearest
integer.
[0271] MDAP (Formic). The HPLC analysis was conducted on an Xselect
CSH C18 column (150 mm.times.30 mm i.d. 5 .mu.m packing diameter)
at ambient temperature, eluting with 0.1% formic acid in water
(Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) using
an elution gradient of between 0 and 100% solvent B over 15 or 25
min.
[0272] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm. The mass spectra were recorded on a Waters ZQ
Mass Spectrometer using alternate-scan positive and negative
electrospray. Ionisation data was rounded to the nearest
integer.
[0273] MDAP (TFA). The HPLC analysis was conducted on an Xselect
CSH C18 column (150 mm.times.30 mm i.d. 5 .mu.m packing diameter)
at ambient temperature, eluting with 0.1% v/v solution of
trifluoroacetic acid in water (Solvent A) and 0.1% v/v solution of
trifluoroacetic acid in acetonitrile (Solvent B) using an elution
gradient of between 0 and 100% solvent B over 15 or 25 min.
[0274] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm. The mass spectra were recorded on a Waters ZQ
Mass Spectrometer using alternate-scan positive and negative
electrospray. Ionisation data was rounded to the nearest
integer.
[0275] NMR
[0276] Spectra were run on either a 400 MHz or 600 MHz NMR machine
at either 302 K or at 392-393 K for VT spectra.
Intermediate 1: tert-Butyl
2-chloro-6-(methylcarbamoyl)isonicotinate
##STR00005##
[0278] 2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane
2,4,6-trioxide (40.7 g, 64.0 mmol) was added to a solution of
4-(tert-butoxycarbonyl)-6-chloropicolinic acid (15 g, 58.2 mmol,
commercially available from, for example, Anichem) and Et.sub.3N
(16.23 mL, 116 mmol) in DCM (100 mL) at rt, then the mixture was
stirred for 20 min before addition of methanamine (2M in THF, 38.8
mL, 78 mmol). The mixture was stirred for 2 h, then washed with
water (100 mL) and saturated sodium bicarbonate solution, then
dried and evaporated in vacuo to give a pale yellow gum. This was
dissolved in DCM and loaded onto a 340 g silica column, then eluted
with 0-40% EtOAc/cyclohexane and the product-containing fractions
were evaporated in vacuo to give tert-butyl
2-chloro-6-(methylcarbamoyl)isonicotinate (6.9 g, 25.5 mmol, 43.8%
yield) as a pale yellow gum which crystallised on standing.
[0279] LCMS (2 min High pH): Rt=1.16 min, [MH].sup.+=271.2.
[0280] .sup.1H NMR (400 MHz, CDCl.sub.3-d) .delta. ppm 8.55 (d,
J=1.2 Hz, 1 H) 7.95 (d, J=1.2 Hz, 1 H) 7.79 (br. s, 1 H) 3.05 (d,
J=4.9 Hz, 3 H) 1.61 (s, 9 H)
Intermediate 2: 4-tert-Butyl 2-ethyl
6-(methylcarbamoyl)pyridine-2,4-dicarboxylate
##STR00006##
[0282] tert-Butyl 2-chloro-6-(methylcarbamoyl)isonicotinate (4.2 g,
15.51 mmol) was dissolved in a mixture of DMF (50 mL) and ethanol
(50 mL), then triethylamine (4.71 g, 46.5 mmol) and
[(R)-(+)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl]palladium(II)
chloride (0.621 g, 0.78 mmol) were added and the mixture was purged
with carbon monoxide, then sealed and a balloon full of carbon
monoxide fitted. The mixture was heated at 70.degree. C. over the
weekend, then evaporated in vacuo and the residue was partitioned
between water (100 mL) and EtOAc (100 mL). The organic layer was
washed with water (100 mL), dried and evaporated in vacuo. The dark
brown residue was purified by chromatography on a 100 g silica
column eluting with 0-50% EtOAc/cyclohexane to give 4-tert-butyl
2-ethyl 6-(methylcarbamoyl)pyridine-2,4-dicarboxylate (4.2 g, 13.62
mmol, 88% yield) as a pale yellow gum.
[0283] LCMS (2 min High pH): Rt=1.11 min, [MH]+=309.2.
[0284] .sup.1H NMR (400 MHz, CDCl.sub.3-d) .delta. ppm 8.80 (d,
J=1.5 Hz, 1 H), 8.67 (d, J=1.7 Hz, 1 H), 8.08 (br. d, J=3.4 Hz, 1
H), 4.50 (q, J=7.1 Hz, 2 H), 3.08 (d, J=5.1 Hz, 3 H), 1.63 (s, 9
H), 1.46 (t, J=7.1 Hz, 3 H)
Intermediate 3: tert-Butyl
2-(hydroxymethyl)-6-(methylcarbamoyl)isonicotinate
##STR00007##
[0286] Calcium chloride (4.54 g, 40.9 mmol) was added to a solution
of 4-tert-butyl 2-ethyl
6-(methylcarbamoyl)pyridine-2,4-dicarboxylate (4.2 g, 13.62 mmol)
in a mixture of ethanol (50 mL) and 2-MeTHF (50 mL) at 0.degree.
C., then sodium tetrahydroborate (0.773 g, 20.43 mmol) was added
and the resulting red mixture was stirred for 2 h allowing the
mixture to warm to rt. The mixture was allowed to stand overnight,
then cooled in an ice bath and ammonium chloride solution (100 mL)
was added slowly over 20 min. The mixture was extracted with EtOAc
(2.times.150 mL), then the organics were dried and evaporated in
vacuo and the residue purified by chromatography on a 50 g silica
column to give tert-butyl
2-(hydroxymethyl)-6-(methylcarbamoyl)isonicotinate (2.2 g, 8.26
mmol, 61% yield) as a beige solid.
[0287] LCMS (2 min High pH): Rt=0.84 min, [MH]+=267.3.
[0288] .sup.1H NMR (400 MHz, CDCl.sub.3-d) .delta. ppm 8.49-8.58
(m, 1 H), 7.90-8.02 (m, 2 H), 4.87 (s, 2 H), 3.05 (d, J=5.1 Hz, 3
H), 1.61 (s, 9 H). 1 exchangeable proton not observed.
Intermediate 4: tert-Butyl
2-(chloromethyl)-6-(methylcarbamoyl)isonicotinate
##STR00008##
[0290] tert-Butyl
2-(hydroxymethyl)-6-(methylcarbamoyl)isonicotinate (1.5 g, 5.63
mmol) was dissolved in DCM (5 mL), sulfurous dichloride (1.26 mL,
16.90 mmol) was added and the reaction stirred at rt for 4 h, then
the mixture was quenched by the addition of saturated sodium
bicarbonate solution and the mixture was stirred for 20 min, then
the organic layer was separated, dried and evaporated in vacuo to
give tert-butyl 2-(chloromethyl)-6-(methylcarbamoyl)isonicotinate
(1.35 g, 4.74 mmol, 84% yield) as a colourless solid.
[0291] LCMS (2 min High pH): Rt=1.13 min, [MH]+=285.2.
[0292] .sup.1H NMR (400 MHz, CDCl.sub.3-d) .delta. ppm 8.59 (d,
J=1.2 Hz, 1 H) 8.11 (d, J=1.2 Hz, 1 H) 7.95 (br. s., 1 H) 4.72 (s,
2 H) 3.07 (d, J=5.1 Hz, 3 H) 1.62 (s, 9 H)
Intermediate 5: tert-Butyl
2-formyl-6-(methylcarbamoyl)isonicotinate
##STR00009##
[0294] tert-Butyl
2-(hydroxymethyl)-6-(methylcarbamoyl)isonicotinate (543 mg, 2.04
mmol) was dissolved in DCM (5 mL). Dess-Martin periodinane (1009
mg, 2.38 mmol) was added and the mixture stirred at rt for 3 h.
Sodium thiosulfate was added to the reaction mixture then
NaHCO.sub.3 was also added. The resultant mixture was stirred for
15 min. The aqueous phase was extracted with DCM thee times and the
combined organic layers were dried over MgSO.sub.4 and evaporated.
The crude product was purified by chromatography on SiO.sub.2
(Biotage SNAP 10 g, eluting with 0-50% ethyl acetate/cyclohexane).
The desired fractions were concentrated to give tert-butyl
2-formyl-6-(methylcarbamoyl)isonicotinate (501 mg, 1.71 mmol, 84%
yield) as a colourless oil.
[0295] LCMS (2 min Formic): Rt=0.97 min, [MH].sup.+=265.3.
[0296] .sup.1H NMR (400 MHz, CDCl.sub.3-d) .delta. ppm 10.14 (s, 1
H), 8.88 (d, J=1.5 Hz, 1 H), 8.55 (d, J=1.5 Hz, 1 H), 8.00 (br. s.,
1 H), 3.12 (d, J=4.9 Hz, 3 H), 1.62-1.66 (m, 9 H)
Intermediate 6: tert-Butyl
2-benzyl-6-(methylcarbamoyl)isonicotinate
##STR00010##
[0298] tert-Butyl 2-chloro-6-(methylcarbamoyl)isonicotinate (5 g,
18.47 mmol, commercially available from, for example, Anichem) and
PdCl.sub.2(PPh.sub.3).sub.2 (1.296 g, 1.85 mmol) were dissolved in
THF (50 mL) and benzylzinc(II) bromide (0.5M in THF, 55.4 mL, 27.7
mmol) was added, then the mixture was heated at 70.degree. C. for 2
h. The solvent was evaporated in vacuo and the residue purified by
chromatography on a 100 g silica column eluting with 0-50%
EtOAc/cyclohexane to give tert-butyl
2-benzyl-6-(methylcarbamoyl)isonicotinate (5.7 g, 17.46 mmol, 95%
yield) as a dark brown oil which was used in the next step without
further purification.
[0299] LCMS (2 min High pH): Rt=1.30 min, [MH].sup.+=327.3.
[0300] 1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.46 (d, J=1.2 Hz,
1 H), 7.91-8.07 (m, 1 H), 7.78 (d, J=1.2 Hz, 1 H), 7.28-7.34 (m, 2
H), 7.21-7.27 (m, 3 H), 4.21 (s, 2 H) 3.05 (d, J=5.1 Hz, 3 H), 1.58
(s, 9 H)
Intermediate 7: 2-Benzyl-6-(methylcarbamoyl)isonicotinic acid
##STR00011##
[0302] tert-Butyl 2-benzyl-6-(methylcarbamoyl)isonicotinate (2.5 g,
7.66 mmol) was dissolved in DCM (30 mL), then TFA (10 mL, 130 mmol)
was added and the mixture was stirred for 3 h at rt. The solvent
was evaporated in vacuo to give a pale yellow gum. The crude
material was dissolved in DCM (100 mL) and washed with water (100
mL), the organic layer was dried and evaporated in vacuo to give
2-benzyl-6-(methylcarbamoyl)isonicotinic acid (2.0 g, 7.40 mmol,
97% yield) as a pale yellow solid
[0303] LCMS (2 min High pH): Rt=0.63 min, [MH].sup.+=271.3.
[0304] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 13.76 (br. s., 1
H), 8.73 (d, J=4.9 Hz, 1 H), 8.24 (d, J=1.5 Hz, 1 H), 7.83 (d,
J=1.5 Hz, 1 H), 7.34-7.39 (m, 2 H), 7.28-7.34 (m, 2 H) 7.19-7.25
(m, 1 H), 4.26 (s, 2 H), 2.87 (d, J=4.6 Hz, 3 H)
Intermediate 8: (+/-)-tert-Butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate
##STR00012##
[0306] tert-Butyl 2-chloro-6-(methylcarbamoyl)isonicotinate (0.5 g,
1.85 mmol) was dissolved in THF (20 mL) and palladium dichloride
bis triphenylphosphine (0.130 g, 0.19 mmol) was added. The solution
was sparged with nitrogen for 5 min, then (1-phenylethyl)zinc(II)
bromide (0.5M in THF, 7.39 mL, 3.69 mmol, commercially available
from, for example, Sigma Aldrich) was added and the mixture heated
at 70.degree. C. for 2 h. The solution was diluted with EtOAc (100
mL) and washed with water (100 mL), dried and evaporated in vacuo.
The residue was purified by chromatography on a 25 g silica column
eluting with 0-50% EtOAc/cyclohexane and the product-containing
fractions evaporated in vacuo to give tert-butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate (0.41 g, 1.20
mmol, 65% yield) as a dark yellow oil.
[0307] LCMS (2 min High pH): Rt=1.37 min, [MH].sup.+=341.3.
[0308] .sup.1H NMR (400 MHz, CDCl.sub.3-d) .delta. ppm 8.45 (d,
J=1.5 Hz, 1 H), 8.02 (br. s., 1 H), 7.81 (d, J=1.2 Hz, 1 H),
7.18-7.36 (obs. m, 5 H), 4.38 (q, J=7.3 Hz, 1 H), 3.07 (d, J=5.1
Hz, 3 H), 1.74 (d, J=7.3 Hz, 3 H), 1.59 (s, 9 H)
Intermediate 9:
(+/-)-2-(Methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid
##STR00013##
[0310] tert-Butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate (0.41 g, 1.20
mmol) was dissolved in TFA (6 mL) and stirred for 3 h at rt, then
the mixture was evaporated in vacuo and the residue partitioned
between water (20 mL) and DCM (20 mL). The organic layer was dried
and evaporated in vacuo to give
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid (305 mg,
1.07 mmol, 89% yield) as a grey foam.
[0311] LCMS (2 min High pH): Rt=0.69 min, [MH].sup.+=285.2.
[0312] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 13.74 (br. s., 1
H), 8.75 (m, J=4.9 Hz, 1 H), 8.21 (d, J=1.5 Hz, 1 H,) 7.82 (d,
J=1.5 Hz, 1 H), 7.42 (br. d, J=7.1 Hz, 2 H), 7.30 (t, J=7.5 Hz, 2
H), 7.16-7.23 (m, 1 H), 4.47 (q, J=7.1 Hz, 1 H), 2.89 (d, J=4.9 Hz,
3 H), 1.72 (d, J=7.3 Hz, 3 H)
Intermediate 10: (R)-tert-Butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate
Intermediate 11: (S)-tert-Butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate
##STR00014##
[0314] (+/-)-tert-Butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate (7.78 g) was
purified by chiral HPLC. The racemate was dissolved in EtOH (150
mL). Injection: 1.1 mL of the solution was injected via preparative
autosampler, onto the column (20% EtOH/heptane+0.2% isopropylamine,
flow rate=42.5 mL/min, detection wavelength=280 nm, band width 140
nm, reference 400 nm bandwidth 100 nm, Column 30 mm.times.25 cm
Chiralcel OJ-H). Fractions from 11.2-13.7 min were bulked and
labelled peak 1. Fractions from 15.7-19 min were bulked and
labelled peak 2. The bulked fractions were concentrated in vacuo
and then transferred to weighed flasks.
[0315] The fractions corresponding to peak 1 were collected to
afford intermediate 10 (2.84 g)
[0316] LCMS (2 min High pH): Rt=1.35 min, [MH].sup.+=341.3
[0317] The fractions corresponding to peak 2 were collected to
afford intermediate 11 (2.80 g)
[0318] LCMS (2 min High pH): Rt=1.35 min, [MH].sup.+=341.3
[0319] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.46 (d, J=1.2
Hz, 1 H), 8.03 (br. s., 1 H), 7.82 (d, J=1.5 Hz, 1 H), 7.20-7.36
(m, 5 H), 4.39 (q, J=7.2 Hz, 1 H), 3.08 (d, J=5.1 Hz, 3 H), 1.76
(d, J=7.1 Hz, 3 H), 1.60 (s, 9 H)
Intermediate 12:
(S)-2-(Methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid
##STR00015##
[0321] A mixture of (S)-tert-butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate (2.19 g, 6.43
mmol, intermediate 11) and trifluoroacetic acid (10 mL, 130 mmol)
in DCM (15 mL) was stirred at rt for 19 h. The volatiles were
evaporated from the mixture in vacuo and the oily residue
redissolved in acetonitrile (ca. 10 mL) and the solvent evaporated
in vacuo. The orange oily residue had ether (ca. 10 mL) added and a
white solid precipitated. The solid was filtered, washed with ether
(2.times.5 mL) and dried in vacuo to give the desired product as a
white solid; (S)-2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic
acid (1.18 g, 4.14 mmol, 64% yield)
[0322] The solvent from the mother liquor of the second ether wash
was evaporated under a stream of nitrogen to give a second batch of
the desired product as a white solid;
(S)-2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid (95.6
mg, 0.336 mmol, 5.23% yield)
[0323] The solvent from the combined mother liquors of the initial
trituration and first ether wash were evaporated under a stream of
nitrogen and the orange viscous oil which resulted was triturated
with ether (5 mL). The mother liquor was decanted away and the
solid triturated with further ether (3.times.5 mL), each time
decanting the mother liquor. The solid was dried in vacuo to give a
third batch of the desired product as a cream solid, yield;
(S)-2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid (310.8
mg, 1.09 mmol, 17% yield)
[0324] The combined mother liquors from the isolation of the above
batch were evaporated under a stream of nitrogen and the resultant
orange semi-crystalline solid was washed with ether (3 mL). The
mother liquor was decanted away and the solid triturated with
further ether (3.times.3 mL), each time decanting the mother
liquor. The solid was dried in vacuo to give a fourth batch of the
desired product as a cream solid (100.4 mg)
[0325] Total product isolated summed over the four batches=1.68 g,
92%.
[0326] LCMS (2 min Formic): Rt=1.00 min, [MH].sup.+=285.3
[0327] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 13.81 (br. s., 1
H), 8.80 (q, J=4.5 Hz, 1 H), 8.22 (s, 1 H), 7.83 (d, J=1.5 Hz, 1
H), 7.43 (d, J=7.1 Hz, 2 H), 7.27-7.34 (m, 2 H), 7.16-7.24 (m, 1
H), 4.48 (q, J=7.3 Hz, 1 H), 2.90 (d, J=4.9 Hz, 3 H), 1.73 (d,
J=7.3 Hz, 3 H)
Intermediate 13:
(R)-2-(Methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid
##STR00016##
[0329] (R)-tert-Butyl
2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinate (497 mg, 1.46
mmol, intermediate 10) was taken up in DCM (5 mL), TFA (0.5 mL,
6.49 mmol) was added and the reaction left to stir at rt overnight.
TFA (0.5 mL, 6.49 mmol) was added again and the reaction was
refluxed at 50.degree. C. for 3 h. More TFA (1 mL) was added to the
reaction, which was then left to stir for a further 2 h. The
reaction was concentrated in vacuo. The sample was loaded in
methanol and purified by SPE on sulphonic acid (SCX, 2 g) and
eluted through with methanol. The appropriate fractions were
combined and evaporated in vacuo to give the required product (350
mg) as a pink solid.
[0330] LCMS (2 min High pH): Rt=0.68 min, [MH].sup.+=285.2.
Intermediate 14: tert-Butyl
2-((1H-indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinate
##STR00017##
[0332] tert-Butyl 2-(chloromethyl)-6-(methylcarbamoyl)isonicotinate
(100 mg, 0.35 mmol) was combined with (1H-indol-4-yl)boronic acid
(113 mg, 0.70 mmol), potassium carbonate (291 mg, 2.107 mmol) and
PdCl.sub.2(dppf) (51.4 mg, 0.07 mmol) in 1,4-dioxane (1 mL) and
water (0.5 mL) in a 2 mL microwave vial. This was heated at
120.degree. C. for 40 min. The solution was filtered though celite
eluting with EtOAc (10 mL) then dried and concentrated. The crude
product was purified by chromatography on SiO.sub.2 (Biotage SNAP
10 g, eluting with 0-60% ethyl acetate/cyclohexane). The desired
fractions were concentrated to give tert-butyl
2-((1H-indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinate (75.4
mg, 0.17 mmol, 47% yield) as a white solid.
[0333] LCMS (2 min Formic): Rt=1.20 min, [MH]+=366.2.
[0334] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 8.30 (d, J=1.2
Hz, 1 H), 7.76 (d, J=1.2 Hz, 1 H), 7.31 (d, J=8.3 Hz, 1 H), 7.21
(d, J=3.2 Hz, 1 H), 7.03-7.11 (m, 1 H), 6.91 (br. d, J=7.1 Hz, 1
H), 6.47 (dd, J=3.2, 0.7 Hz, 1 H), 4.52 (s, 2 H) 2.99 (s, 3 H),
1.54 (s, 9 H). Exchangeables not observed.
Intermediate 15:
2-((1H-Indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinic acid
##STR00018##
[0336] To a solution of tert-butyl
2-((1H-indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinate (75.4
mg, 0.17 mmol) in DCM (3 mL) was added TFA (0.60 mL, 7.79 mmol) and
the reaction mixture was stirred at rt overnight. Further TFA (0.3
mL, 0.17 mmol) was added and the resultant mixture stirred for 3 h.
The reaction mixture was concentrated in vacuo to give
2-((1H-indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinic acid (184
mg, 0.15 mmol, 90% yield, .about.25% purity).
[0337] LCMS (2 min Formic): Rt=0.88 min, [MH]+=310.1.
[0338] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 11.59-12.89
(m, 1 H), 11.11 (br. s., 1 H), 8.76 (d, J=4.9 Hz, 1 H), 8.19 (d,
J=1.2 Hz, 1 H), 7.71 (d, J=1.5 Hz, 1 H), 7.21-7.39 (m, 2 H), 7.05
(t, J=7.6 Hz, 1 H), 6.95 (d, J=6.8 Hz, 1 H), 6.46-6.56 (m, 1 H),
4.48 (s, 2 H), 2.88 (d, J=4.9 Hz, 3 H).
Intermediate 16: (+/-)-tert-Butyl
2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate
##STR00019##
[0340] To a solution of tert-butyl
2-formyl-6-(methylcarbamoyl)isonicotinate (118 mg, 0.45 mmol) in
THF (1.5 mL) at 0.degree. C., was added dropwise phenylmagnesium
bromide (1M in THF, 2 mL, 2 mmol). The reaction mixture was stirred
for 2 h. The reaction mixture was poured onto a saturated ammonium
chloride aqueous solution and extracted with EtOAc (20 mL.times.3).
The organic layer was dried over MgSO.sub.4 and concentrated in
vacuo. The crude product was purified by chromatography on
SiO.sub.2 (Biotage SNAP 10 g, eluting with 0-60% ethyl
acetate/cyclohexane). The desired fractions were concentrated to
give tert-butyl
2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (43 mg,
0.11 mmol, 24% yield).
[0341] LCMS (2 min Formic): Rt=1.09 min, [MH].sup.+=343.3.
[0342] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 8.38 (d, J=1.2
Hz, 1 H) 8.05 (d, J=1.2 Hz, 1 H) 7.42-7.47 (m, 2 H) 7.22-7.36 (m, 3
H) 5.95 (s, 1 H) 2.99 (s, 3 H) 1.60 (s, 9 H). Exchangeables not
observed.
Intermediate 17:
(+/-)-2-(Hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic
acid
##STR00020##
[0344] To a solution of tert-butyl
2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (43 mg,
0.13 mmol) in DCM (0.5 mL) was added TFA (0.4 mL, 5.19 mmol) and
the reaction mixture was stirred for 2 h and then overnight.
Further TFA (0.4 mL, 0.13 mmol) was added and the reaction mixture
was stirred for 5 h, then the solvent was removed to give
2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic acid
(47.9 mg, 0.12 mmol, 93% yield, 70% purity) which was used directly
in the next step.
[0345] LCMS (2 min Formic): Rt=0.74 min, [MH].sup.+=287.1.
[0346] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 8.45 (d, J=1.2
Hz, 1 H), 8.10 (d, J=1.5 Hz, 1 H), 7.41-7.48 (m, 2 H), 7.21-7.38
(m, 3 H), 5.97 (s, 1 H), 2.99 (s, 3 H). Exchangeables not
observed.
Intermediate 18: (+/-)-tert-Butyl
2-(chloro(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate
##STR00021##
[0348] To a solution of tert-butyl
2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (46 mg,
0.13 mmol) in DCM (4 mL) at 0.degree. C., was added dropwise
thionyl chloride (30 .mu.L, 0.41 mmol). The reaction mixture was
then stirred at rt for 12 h. Further thionyl chloride (50 .mu.L,
0.69 mmol) was added and the resultant mixture was stirred for 5 h
then concentrated in vacuo to give tert-butyl
2-(chloro(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (54 mg)
which was used without purification in the subsequent reaction.
[0349] LCMS (2 min Formic): Rt=1.33 min, [MH].sup.+=361.1
Intermediate 19: (+/-)-tert-Butyl
2-(methoxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate
##STR00022##
[0351] A solution of tert-butyl
2-(chloro(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (54 mg,
0.15 mmol) in methanol (5 mL) was stirred over the weekend. The
reaction mixture was then heated under reflux for 1 h initially,
then 4 h and finally overnight. The reaction mixture was then
concentrated in vacuo. The resultant crude product was purified by
flash silica chromatography (SNAP 10 g cartridge, eluent: 0-50%
ethyl acetate/cyclohexane). The desired fractions were combined and
concentrated in vacuo to give tert-butyl
2-(methoxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (33 mg,
0.08 mmol, 56% yield) as a colourless oil.
[0352] LCMS (2 min Formic): Rt=1.26 min, [MH].sup.+=357.2.
[0353] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 8.38 (d, J=1.5
Hz, 1 H), 8.10 (d, J=1.5 Hz, 1 H), 7.44 (d, J=7.3 Hz, 2 H),
7.30-7.38 (m, 2 H) 7.23-7.30 (m, 1 H), 5.54 (s, 1 H), 3.44 (s, 3
H), 2.98 (s, 3 H), 1.61 (s, 9 H). Exchangeable proton not
observed.
Intermediate 20:
(+/-)-2-(Methoxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic
acid
##STR00023##
[0355] To a solution of tert-butyl
2-(methoxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinate (33 mg,
0.09 mmol) in DCM (1 mL) was added 2,2,2-trifluoroacetic acid (0.5
mL, 6.49 mmol) and the reaction mixture was stirred overnight. This
was then washed with water and extracted with DCM three times, then
it was dried. The solvent was removed in vacuo to give
2-(methoxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic acid
(44.9 mg, 0.09 mmol, 97% yield, .about.60% purity)
[0356] LCMS (2 min Formic): Rt=0.91 min, [MH].sup.+=301.1
[0357] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 8.45 (d, J=1.2
Hz, 1 H), 8.17 (d, J=1.5 Hz, 1 H), 7.98 (br. s, 1 H), 7.42-7.49 (m,
2 H), 7.31-7.38 (m, 2 H), 7.27 (m, J=7.3 Hz, 1 H), 5.55 (s, 1 H)
3.45 (s, 3 H), 2.99 (d, J=3.2 Hz, 3 H). 1 exchangeable proton not
observed.
EXAMPLES
Example 1:
6-Benzyl-N.sup.2-methyl-N.sup.4-propylpyridine-2,4-dicarboxamid-
e
##STR00024##
[0359] 2-Benzyl-6-(methylcarbamoyl)isonicotinic acid (50 mg, 0.19
mmol), HATU (105 mg, 0.28 mmol), DIPEA (0.1 mL, 0.57 mmol),
propan-1-amine (0.03 mL, 0.37 mmol) and DMF (1 mL) were stirred at
rt under N.sub.2. After stirring for 45 min the solution was
concentrated to give an orange oil (250 mg). This was purified by
chromatography on SiO.sub.2 (Biotage SNAP 25 g cartridge, eluting
with 30-100% ethylacetate/cyclohexane. The appropriate fractions
were concentrated to give
6-benzyl-N.sup.2-methyl-N.sup.4-propylpyridine-2,4-dicarboxamide
(28 mg, 0.081 mmol, 44% yield) as a colourless oil. LCMS (2 min
Formic): Rt=0.98 min, [MH]+=312.0.
[0360] The following examples were prepared in a similar manner to
example 1 from intermediate 7,
2-benzyl-6-(methylcarbamoyl)isonicotinic acid and the appropriate
commercially available amine monomer, to provide the listed
examples.
Examples
TABLE-US-00004 [0361] Mass Yield Rt Ex No. Name Structure (mg) (%)
[MH].sup.+ (min) 2 6-Benzyl-N.sup.2,N.sup.4- dimethylpyridine- 2,4-
dicarboxamide ##STR00025## 74.8 89 284.1 0.83 (formic) 3
(+/-)-6-Benzyl- N.sup.4-(3- hydroxybutyl)- N.sup.2- methylpyridine-
2,4- dicarboxamide ##STR00026## 10.6 28 342 0.83 (High pH) 4
N.sup.4-(2-(1H- Imidazol-5- yl)ethyl)-6- benzyl-N.sup.2-
methylpyridine- 2,4- dicarboxamide ##STR00027## 10.5 26 364 0.56
(High pH) 5 6-Benzyl-N.sup.4-(3- hydroxypropyl)- N.sup.2-
methylpyridine- 2,4- dicarboxamide ##STR00028## 9.1 25 328 0.79
(High pH) 7 6-Benzyl-N.sup.4-(3- methoxypropyl)- N.sup.2-
methylpyridine- 2,4- dicarboxamide ##STR00029## 11.7 31 342 0.91
(High pH) 8 N.sup.4-(2-(1H-Pyrazol- 3-yl)ethyl)-6- benzyl-N.sup.2-
methylpyridine- 2,4- dicarboxamide ##STR00030## 11.2 25 364 0.82
(High pH) 9 6-Benzyl-N.sup.4- isopentyl-N.sup.2- methylpyridine-
2,4- dicarboxamide ##STR00031## 61.6 90 340.3 1.15 (High pH) 10
6-Benzyl-N.sup.2- methyl-N.sup.4- (oxazol-2- yl)pyridine-2,4-
dicarboxamide ##STR00032## 40.3 61 337.2 0.87 (formic) 13
6-Benzyl-N.sup.2- methyl-N.sup.4-(1H- pyrazol-5- yl)pyridine-2,4-
dicarboxamide ##STR00033## 23.1 36 336.3 0.88 (High pH) 20
N.sup.4-((1H-Pyrazol-3- yl)methyl)-6- benzyl-N.sup.2-
methylpyridine- 2,4- dicarboxamide ##STR00034## 8.7 13 350.2 0.83
(formic) 24 6-Benzyl-N.sup.2- methyl-N.sup.4-((1- methyl-1H-
pyrazol-4- yl)methyl)pyridine- 2,4- dicarboxamide ##STR00035## 80
57 364.3 0.88 (high pH) 28 6-Benzyl-N.sup.2- methyl-N.sup.4-(1-
methyl-1H- pyrazol-3- yl)pyridine-2,4- dicarboxamide ##STR00036##
63.8 91 350.3 0.94 (high pH) 30 6-Benzyl-N.sup.4-(3,3-
diethoxypropyl)- N.sup.2- methylpyridine- 2,4- dicarboxamide
##STR00037## 91.3 79 400.4 1.08 (High pH) 34 6-Benzyl-N.sup.4-(4,4-
diethoxybutyl)- N.sup.2- methylpyridine- 2,4- dicarboxamide
##STR00038## 651 82 368.3 1.08 (formic) 51 6-Benzyl-N.sup.4-(1-(2-
hydroxyethyl)- 1H-pyrazol-3-yl)- N.sup.2- methylpyridine- 2,4-
dicarboxamide ##STR00039## 40.5 56 380.4 0.86 (high pH)
Example 6:
(+/-)-N.sup.4-(2-Hydroxyethyl)-N.sup.2-methyl-6-(1-phenylethyl)-
pyridine-2,4-dicarboxamide
##STR00040##
[0363] 2-(Methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid (50
mg, 0.18 mmol) was taken up in DMF (2 mL), DIPEA (0.092 mL, 0.53
mmol) was added, shortly followed by HATU (100 mg, 0.26 mmol) and
the reaction left to stir at rt for 10 min. 2-Aminoethanol (0.011
mL, 0.18 mmol) was added and the reaction was left to stir for a
further 1 h. The mixture was concentrated in vacuo. The residue was
dissolved in ethyl acetate (10 mL) before being washed with sodium
bicarbonate solution (10 mL). The aqueous layer was washed with
ethyl acetate (10 mL). The combined organic layers were then washed
with brine (10 mL) before being dried over sodium sulfate and
filtered through a hydrophobic frit. The reaction was then
concentrated in vacuo. The crude product was dissolved in 1:1
MeOH:DMSO (0.8 mL) and purified by MDAP (high pH). The MDAP failed
to collect the products and the product was instead collected with
the waste solvent. The waste material was evaporated in vacuo. The
crude product was dissolved in DMSO and 1:1 DMSO:MeOH (0.3 mL) and
purified by MDAP (high pH). The solvent was evaporated in vacuo to
give the title compound (5.9 mg).
[0364] LCMS (2 min High pH): Rt=0.87 min, [MH]+=328.2.
Example 11:
6-Benzyl-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyrazol-4-yl)pyridine-2,4-di-
carboxamide
##STR00041##
[0366] To a mixture of 1-methyl-1H-pyrazol-4-amine, hydrochloride
(33.0 mg, 0.25 mmol), HATU (92.8 mg, 0.24 mmol) and DIPEA (0.100
mL, 0.57 mmol) in DMF (1 mL) was added
2-benzyl-6-(methylcarbamoyl)isonicotinic acid (50.9 mg, 0.19 mmol).
This mixture was stirred at rt for 30 min. The mixture was
evaporated under a stream of nitrogen and the resulting sticky dark
brown solid redissolved in DMSO (2 mL) and directly purified by
MDAP (2.times.1 mL injection, high pH). The required fractions
(fraction 1 for both runs) were evaporated under a stream of
nitrogen, redissolved in methanol (approx. 2 mL) and
dichloromethane (approx. 2 mL) and combined. This solution was
evaporated under a stream of nitrogen and the residue dried in
vacuo to give the desired product as a light pink glassy
solid--6-benzyl-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyrazol-4-yl)p-
yridine-2,4-dicarboxamide (51.9 mg, 0.15 mmol, 79% yield).
[0367] LCMS (2 min High pH): Rt=0.91 min, [MH]+=350.3.
[0368] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 10.89 (s, 1
H) 8.78 (br. q, J=4.6, 4.6, 4.6 Hz, 1 H), 8.39 (s, 1 H), 8.04 (s, 1
H), 7.90 (d, J=0.7 Hz, 1 H), 7.59 (s, 1 H) 7.38 (br. d, J=6.8 Hz, 2
H), 7.32 (t, J=7.5 Hz, 2 H), 7.22 (br. t, J=7.3, 7.3 Hz, 1 H), 4.25
(s, 2 H) 3.82 (s, 3 H), 2.88 (d, J=4.9 Hz, 3 H)
Example 12:
6-Benzyl-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,4-dicarboxami-
de
##STR00042##
[0370] To a mixture of 2-benzyl-6-(methylcarbamoyl)isonicotinic
acid (251.8 mg, 0.93 mmol) and HATU (565.5 mg, 1.49 mmol) was added
1H-pyrazol-4-amine hydrochloride (166.0 mg, 1.39 mmol) and DMF (4
mL). DIPEA (0.570 mL, 3.26 mmol) was added and the mixture was
stirred at rt for 3.5 h. The mixture was concentrated under a
stream of nitrogen and then diluted with acetonitrile to a total
volume of 5 mL and directly purified by MDAP (5.times.1 mL
injection; high pH) and the required fractions (fraction 1 from
each run) were evaporated under a stream of nitrogen. The residues
were each redissolved in methanol (.about.5 mL), combined into a
tarred vial and the solvent evaporated under a stream of nitrogen
to give a pale yellow
solid--6-benzyl-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,4-dica-
rboxamide (198.9 mg, 0.59 mmol, 64% yield)
[0371] LCMS (2 min High pH): Rt=0.85 min, [MH]+=336.1.
[0372] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 12.71 (br. s., 1
H), 10.88 (s, 1 H), 8.78 (q, J=4.5 Hz, 1 H), 8.40 (d, J=1.5 Hz, 1
H), 8.02 (br. s., 1 H), 7.90 (d, J=1.5 Hz, 1 H), 7.70 (br. s., 1
H), 7.36-7.41 (m, 2 H), 7.29-7.36 (m, 2 H), 7.19-7.26 (m, 1 H),
4.25 (s, 2 H), 2.88 (d, J=4.9 Hz, 3 H)
Example 80:
6-Benzyl-N.sup.4-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)-N.sup.2-m-
ethylpyridine-2,4-dicarboxamide
##STR00043##
[0374] To a sealed microwave vial containing sodium hydride (60%
dispersion in mineral oils, 14.9 mg, 0.37 mmol) and
6-benzyl-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,4-dicarboxami-
de (58.9 mg, 0.18 mmol) was added DMF (1 mL). The mixture was
stirred under an atmosphere of nitrogen at rt for 30 min before
2,2-dimethyloxirane (0.019 mL, 0.21 mmol) was added and the mixture
stirred at rt for 35 min. The microwave cap was replaced with a new
one and the mixture heated in a microwave reactor at 60.degree. C.
for 30 min. The microwave cap was replaced with a new one and the
mixture heated in a microwave reactor at 80.degree. C. for 30 min.
Methanol (0.5 mL) was added to quench the reaction and the
resulting orange solution was directly purified by MDAP (2.times.1
mL injection, formic). The required fractions (fraction 1 from both
runs) were evaporated under a stream of nitrogen, the residues were
dissolved in dichloromethane (.about.10 mL), combined and
transferred to a tarred vial before the solvent was evaporated
under a stream of nitrogen and dried in vacuo to give the desired
product as a colourless glass,
6-benzyl-N.sup.4-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)-N.sup.2-m-
ethylpyridine-2,4-dicarboxamide (14.0 mg, 0.03 mmol, 20% yield)
[0375] LCMS (2 min High pH): Rt=0.89 min, [MH]+=408.6.
Example 16:
(S)-N.sup.2-Methyl-6-(1-phenylethyl)-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,-
4-dicarboxamide
##STR00044##
[0377] To a mixture of
(S)-2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid (80.5
mg, 0.28 mmol) and HATU (172.0 mg, 0.45 mmol) was added
1H-pyrazol-4-amine hydrochloride (51.6 mg, 0.43 mmol) and DMF (1.8
mL). DIPEA (0.173 mL, 0.99 mmol) was added and the mixture was
stirred at rt for 2 h. The mixture was concentrated under a stream
of nitrogen and diluted with acetonitrile to a total volume of 2 mL
and directly purified by MDAP (2.times.1 mL injection; formic) and
the required fractions (fraction 1 from both runs) were combined
and evaporated in vacuo. The residue was redissolved in methanol
(.about.6 mL) and transferred to a tarred vial, the solvent
evaporated under a stream of nitrogen and the residue dried in
vacuo to give the desired product as a yellow solid,
(S)-N.sup.2-methyl-6-(1-phenylethyl)-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,-
4-dicarboxamide (87.5 mg, 0.25 mmol, 88% yield)
[0378] LCMS (2 min Formic): Rt=0.91 min, [MH]+=350.3.
[0379] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 10.87 (s, 1 H)
8.80 (d, J=4.2 Hz, 1 H), 8.38 (s, 1 H) 7.80-7.96 (m, 3 H), 7.43 (d,
J=7.6 Hz, 2 H), 7.31 (t, J=7.2 Hz, 2 H), 7.13-7.25 (m, 1 H), 4.45
(q, J=6.6 Hz, 2 H), 2.91 (d, J=3.9 Hz, 3 H), 1.74 (d, J=6.8 Hz, 3
H)
[0380] The following examples were prepared in a similar manner to
example 16 from intermediate 12,
(S)-2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid and the
appropriate commercially available amine monomer, to provide the
listed examples.
Examples
TABLE-US-00005 [0381] Mass Yield Rt Ex No. Name Structure (mg) (%)
[MH].sup.+ (min) 14 (S)-N.sup.2-Methyl-N.sup.4- (1-methyl-1H-
pyrazol-4-yl)-6- (1-phenyl- ethyl)pyridine-2,4- dicarboxamide
##STR00045## 55.4 92 364.4 0.98 (high pH) 15 (S)-N.sup.2-Methyl-6-
(1-phenylethyl)- N.sup.4-(1H-pyrazol- 3-yl)pyridine-2,4-
dicarboxamide ##STR00046## 14.9 27 350.3 0.95 (High pH) 17
(S)-N.sup.2-Methyl-6- (1-phenylethyl)- N.sup.4-(2-(pyridin-2-
yl)ethyl)pyridine- 2,4- dicarboxamide ##STR00047## 63.7 89 389.4
1.01 (High pH) 18 (S)-N.sup.2-Methyl-6- (1-phenylethyl)-
N.sup.4-(2-(pyridin-3- yl)ethyl)pyridine- 2,4- dicarboxamide
##STR00048## 56.9 82 389.4 0.98 (High pH) 19 (S)-N.sup.4-(2-(1H-
Pyrazol-4- yl)ethyl)-N.sup.2- methyl-6-(1- phenyl-
ethyl)pyridine-2,4- dicarboxamide ##STR00049## 45.4 68 378.4 0.92
(High pH) 22 (S)-N.sup.2-Methyl-N.sup.4- (2-(1-methyl-1H-
pyrazol-3- yl)ethyl)-6-(1- phenyl- ethyl)pyridine-2,4-
dicarboxamide ##STR00050## 52.8 78 392.4 0.96 (High pH) 23
(S)-N.sup.4-(2-(1H- Pyrazol-5- yl)ethyl)-N.sup.2- methyl-6-(1-
phenyl- ethyl)pyridine-2,4- dicarboxamide ##STR00051## 47 70 378.4
0.93 (High pH) 25 (S)-N.sup.2-Methyl-N.sup.4- (1-methyl-1H-
pyrazol-3-yl)-6- (1-phenyl- ethyl)pyridine-2,4- dicarboxamide
##STR00052## 61.5 98 364.3 0.98 (formic) 26 (S)-N.sup.4-(2-(1H-
1,2,3-Triazol-1- yl)ethyl)-N.sup.2- methyl-6-(1- phenyl-
ethyl)pyridine-2,4- dicarboxamide ##STR00053## 54.9 100 379.4 0.89
(High pH) 27 (S)-N.sup.4-(2-(1H- Pyrazol-1- yl)ethyl)-N.sup.2-
methyl-6-(1- phenyl- ethyl)pyridine-2,4- dicarboxamide ##STR00054##
50.8 89 378.4 0.96 (High pH) 29 (S)-N.sup.4-(2- (Isoxazol-4-
yl)ethyl)-N.sup.2- methyl-6-(1- phenyl- ethyl)pyridine-2,4-
dicarboxamide ##STR00055## 47.9 87 379.4 1.00 (High pH) 31
(S)-N.sup.2-Methyl-6- (1-phenylethyl)- N.sup.4-(pyridazin-4-
yl)pyridine-2,4- dicarboxamide ##STR00056## 22.3 49 362.3 0.90
(formic) 32 (S)-N.sup.2-Methyl-N.sup.4- (1-methyl-1H-
1,2,4-triazol-3- yl)-6-(1- phenyl- ethyl)pyridine-2,4-
dicarboxamide ##STR00057## 14.7 28 365.3 0.84 (formic) 33
(S)-N.sup.2-Methyl-N.sup.4- (2-(4- methylthiazol-5- yl)ethyl)-6-(1-
phenyl- ethyl)pyridine-2,4- dicarboxamide ##STR00058## 30 52 409.4
1.01 (formic) 35 (S)-N.sup.2-Methyl-6- (1-phenylethyl)-
N.sup.4-(2-(thiazol-4- yl)ethyl)pyridine- 2,4- dicarboxamide
##STR00059## 25 52 395.3 0.98 (formic) 39
(S)-N.sup.2-Methyl-N.sup.4- (2-(4-methyl-4H- 1,2,4-triazol-3-
yl)ethyl)-6-(1- phenyl- ethyl)pyridine-2,4- dicarboxamide
##STR00060## 96.5 99 393.4 0.74 (formic) 40
(S)-N.sup.2-Methyl-N.sup.4- (2-(5-methyl- 1,3,4-oxadiazol-
2-yl)ethyl)-6-(1- phenyl- ethyl)pyridine-2,4- dicarboxamide
##STR00061## 35 63 394.4 1.00 (High pH) 41
(S)-N.sup.2-Methyl-N.sup.4- ((1-methyl-1H- pyrazol-3-
yl)methyl)-6-(1- phenyl- ethyl)pyridine-2,4- dicarboxamide
##STR00062## 32 60 378.4 0.96 (High pH) 42 (S)-N.sup.4-(1-(2-
Hydroxyethyl)- 1H-pyrazol-4-yl)- N.sup.2-methyl-6-(1- phenyl-
ethyl)pyridine-2,4- dicarboxamide ##STR00063## 26.8 40 394.4 0.88
(formic) 43 (S)-N.sup.2-Methyl-6- (1-phenylethyl)-
N.sup.4-(pyrimidin-5- yl)pyridine-2,4- dicarboxamide ##STR00064##
23.8 45 362.3 0.97 (High pH) 44 (S)-N.sup.2-Methyl-N.sup.4-
(2-methyl-2H- tetrazol-5-yl)-6- (1- phenyl- ethyl)pyridine-2,4-
dicarboxamide ##STR00065## 15 29 366.3 0.85 (High pH) 47
(S)-N.sup.2-Methyl-N.sup.4- (2-(1-methyl-1H- 1,2,4-triazol-5-
yl)ethyl)-6-(1- phenyl- ethyl)pyridine-2,4- dicarboxamide
##STR00066## 49.9 85 393.4 0.89 (High pH) 50 (S)-N.sup.4-(1-(2-
Hydroxyethyl)- 1H-pyrazol-3-yl)- N.sup.2-methyl-6-(1- phenyl-
ethyl)pyridine-2,4- dicarboxamide ##STR00067## 48.4 70 394.4 0.91
(High pH) 78 (S)-N.sup.2-Methyl-6- (1-phenyl- ethyl)pyridine-2,4-
dicarboxamide ##STR00068## 58 58 284.3 0.88 (High pH)
Example 21:
(R)-N.sup.2-Methyl-6-(1-phenylethyl)-N.sup.4-(1H-pyrazol-4-yl)pyridine-2,-
4-dicarboxamide
##STR00069##
[0383] To a mixture of
(R)-2-(methylcarbamoyl)-6-(1-phenylethyl)isonicotinic acid (53.7
mg, 0.19 mmol), HATU (86.7 mg, 0.23 mmol) and 1H-pyrazol-4-amine,
hydrochloride (28.9 mg, 0.24 mmol) in DMF (1 mL) was added DIPEA
(0.132 mL, 0.76 mmol). The resulting dark grey solution was stirred
at rt for 2.75 h. The reaction mixture was diluted with DMSO (2 mL)
and directly purified by MDAP (3 mL injection, high pH). The
required fractions (fractions 1 and 2) were combined and evaporated
in vacuo to give the desired product as a light yellow
solid--(R)-N.sup.2-methyl-6-(1-phenylethyl)-N.sup.4-(1H-pyrazol-4-yl)pyri-
dine-2,4-dicarboxamide (39.9 mg, 0.11 mmol, 61% yield)
[0384] LCMS (2 min High pH): Rt=0.92 min, [MH]+=350.3.
Example 36:
6-((1H-Indol-4-yl)methyl)-N.sup.2-methyl-N.sup.4-(2-(1-methyl-1H-pyrazol--
4-yl)ethyl)pyridine-2,4-dicarboxamide
##STR00070##
[0386] To a mixture of
2-((1H-indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinic acid
(30.1 mg, 0.10 mmol) and HATU (58.5 mg, 0.15 mmol) was added a
solution of 2-(1-methyl-1H-pyrazol-4-yl)ethanamine (21.9 mg, 0.18
mmol) in DMF (0.8 mL) followed by DIPEA (0.050 mL, 0.29 mmol) and
the mixture was stirred at rt for 20 min before being left to stand
for 15 h. The mixture was then concentrated under a stream of
nitrogen and diluted with acetonitrile to a total volume of 1 mL
and directly purified by MDAP (1.times.1 mL injection; formic) and
the required fraction (fraction 1) was evaporated under a stream of
nitrogen. The residue was redissolved in dichloromethane (.about.6
mL) and methanol (.about.3 mL) and was transferred to a tarred
vial, the solvent evaporated under a stream of nitrogen and the
residue dried in vacuo to give the desired product as a light brown
solid--6-((1H-indol-4-yl)methyl)-N.sup.2-methyl-N.sup.4-(2-(1-methyl-1H-p-
yrazol-4-yl)ethyl)pyridine-2,4-dicarboxamide (25.8 mg, 0.06 mmol,
64% yield)
[0387] LCMS (2 min Formic): Rt=0.82 min, [MH]+=417.4.
[0388] The following examples were prepared in a similar manner to
example 36 from intermediate 15,
2-((1H-indol-4-yl)methyl)-6-(methylcarbamoyl)isonicotinic acid and
the appropriate commercially available amine monomer, to provide
the listed examples.
Examples
TABLE-US-00006 [0389] Mass Yield Rt Ex No. Name Structure (mg) (%)
[MH].sup.+ (min) 37 6-((1H-Indol-4- yl)methyl)-N.sup.4-(2-
(1H-pyrazol-3- yl)ethyl)-N.sup.2- methylpyridine- 2,4-
dicarboxamide ##STR00071## 21.4 56 403.4 0.78 (formic) 38
6-((1H-Indol-4- yl)methyl)-N.sup.2- methyl-N.sup.4-(1- methyl-1H-
pyrazol-4- yl)pyridine-2,4- dicarboxamide ##STR00072## 15.5 41
389.4 0.84 (formic) 48 6-((1H-Indol-4- yl)methyl)-N.sup.2-
methyl-N.sup.4-(1H- pyrazol-4- yl)pyridine-2,4- dicarboxamide
##STR00073## 34.9 76 375.4 0.81 (High pH) 79 6-((1H-Indol-4-
yl)methyl)-N.sup.2- methylpyridine- 2,4- dicarboxamide ##STR00074##
15 39 309.2 0.75 (Formic)
[0390] Example 45:
(+/-)-6-(Hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyraz-
ol-4-yl)pyridine-2,4-dicarboxamide
##STR00075##
[0391] To a solution of
(.+-.)-2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic
acid (48.1 mg, 0.17 mmol), HATU (94.4 mg, 0.25 mmol) and
1-methyl-1H-pyrazol-4-amine, hydrochloride (35.5 mg, 0.27 mmol) in
DMF (1.0 mL) was added DIPEA (0.117 mL, 0.67 mmol). The resulting
dark orange solution was stirred at rt for 2 h, after which it was
diluted with DMSO (2 mL) and directly purified by MDAP (3 mL
injection, high pH). The required fractions (fractions 1 and 2)
were evaporated under a stream of nitrogen, redissolved in methanol
(2 mL each) and dichloromethane (1 mL each) and combined. This
solution was evaporated under a stream of nitrogen and the residue
dried in vacuo to give the desired product as a light green
solid--(.+-.)-6-(hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1-methyl--
1H-pyrazol-4-yl)pyridine-2,4-dicarboxamide (52.4 mg, 0.14 mmol, 85%
yield).
[0392] LCMS (2 min High pH): Rt=0.77 min, [MH]+=366.3.
[0393] The following examples were prepared in a similar manner to
example 45 from intermediate 17,
(.+-.)-2-(hydroxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic
acid and the appropriate commercially available amine monomer, to
provide the listed examples.
Examples
TABLE-US-00007 [0394] Mass Yield Rt Ex No. Name Structure (mg) (%)
[MH].sup.+ (min) 46 (+/-)-6-(Hydroxy (phenyl)methyl)- N.sup.2-
methyl-N.sup.4-(2-(1- methyl-1H- pyrazol-4- yl)ethyl)pyridine- 2,4-
dicarboxamide ##STR00076## 58.2 88 394.4 0.77 (High pH)
Example 49:
(S*)-6-(Hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(2-(1-methyl-1H-pyr-
azol-4-yl)ethyl)pyridine-2,4-dicarboxamide
Example 77:
(R*)-6-(Hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(2-(1-methyl-1H-pyr-
azol-4-yl)ethyl)pyridine-2,4-dicarboxamide
##STR00077##
[0396]
(+/-)-6-(Hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(2-(1-methyl-
-1H-pyrazol-4-yl)ethyl)pyridine-2,4-dicarboxamide (for a
preparation see Example 46) (52 mg) was purified by chiral HPLC.
The racemate was dissolved in EtOH (2 mL). Injection: 1 mL of the
solution was injected onto the column (30% EtOH/heptane, flow
rate=30 mL/min, detection wavelength=215 nm, 4. Ref 550, 100,
Column 30 mm.times.25 cm Chiralpak IA (5 .mu.m), lot no.
IA11321-01). Total number of injections=7. Fractions from 24-29 min
were bulked and labelled peak 1. Fractions from 29-37 min were
bulked and labelled mix, Fractions from 37-50 min were bulked and
labelled peak 2. The bulked mixed fractions were concentrated in
vacuo and reprocessed using the above method. The bulked pure
fractions were concentrated in vacuo and then transferred to
weighed flasks.
[0397] The fractions corresponding to peak 1 were collected to
afford example 77 (25 mg)
[0398] LCMS (2 min High pH): Rt=0.76 min, [MH]+=394.4.
[0399] The fractions corresponding to peak 2 were collected to
afford example 49 (21 mg)
[0400] LCMS (2 min High pH): Rt=0.76 min, [MH]+=394.4.
Example 52:
(S*)-6-(Hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyrazo-
l-4-yl)pyridine-2,4-dicarboxamide
Example 53:
(R*)-6-(Hydroxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1-methyl-1H-pyrazo-
l-4-yl)pyridine-2,4-dicarboxamide
##STR00078##
[0402] Example 45 (45 mg) was purified by chiral HPLC. The racemate
was dissolved in EtOH (3 mL) with heating. Injection: 1.5 mL of the
solution was injected onto the column (20% EtOH/heptane, flow
rate=20 mL/min, detection wavelength=215 nm, 4. Ref 550, 100,
Column 21.1 mm.times.25 cm (R-R) Whelk O-1 (5 .mu.m), lot no.
#49788). Total number of injections=3. Fractions from 23.5-26 min
were bulked and labelled peak 1. Fractions from 26-28 min were
bulked and labelled mix, Fractions from 28-32 min were bulked and
labelled peak 2. The bulked mixed fractions were concentrated in
vacuo and reprocessed using the above method. The bulked pure
fractions were concentrated in vacuo and then transferred to
weighed flasks.
[0403] The fractions corresponding to peak 1 were collected to
afford example 52 (20 mg)
[0404] LCMS (2 min Formic): Rt=0.75 min, [MH]+=366.3.
[0405] The fractions corresponding to peak 2 were collected to
afford example 53 (20 mg)
[0406] LCMS (2 min Formic): Rt=0.73 min, [MH]+=366.3.
Example 54:
(+/-)-6-(Methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)p-
yridine-2,4-dicarboxamide
##STR00079##
[0408] 2-(Methoxy(phenyl)methyl)-6-(methylcarbamoyl)isonicotinic
acid (150 mg, 0.50 mmol) was added to a dry flask. DMF (2 mL) was
added, followed by HATU (228 mg, 0.60 mmol) and 1H-pyrazol-4-amine,
hydrochloride (59.7 mg, 0.50 mmol). The reaction was stirred for 2
min and then DIPEA (0.262 mL, 1.498 mmol) was added. The reaction
was stirred at rt for 30 min. The reaction mixture was added
directly to 3.times. LCMS vials, diluting with DMSO/MeOH and
purified by 3.times. MDAP (High pH). The appropriate fractions were
concentrated in vacuo to afford the desired product as a yellow
solid--(+/-)-6-(methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H--
pyrazol-4-yl)pyridine-2,4-dicarboxamide (100 mg, 0.27 mmol, 55%
yield)
[0409] LCMS (2 min Formic): Rt=0.82 min, [MH]+=366.3.
[0410] .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 12.69 (br. s., 1
H) 10.92 (s, 1 H), 8.67 (q, J=4.8 Hz, 1 H), 8.45 (d, J=1.7 Hz, 1
H), 8.17 (d, J=1.5 Hz, 1 H), 8.04 (br. s., 1 H), 7.72 (br. s., 1
H), 7.51 (d, J=7.1 Hz, 2 H), 7.36 (t, J=7.5 Hz, 2 H), 7.24-7.31 (m,
1 H), 5.52 (s, 1 H), 3.39 (s, 3 H), 2.88 (d, J=4.9 Hz, 3 H)
Example 55:
(S*)-6-(Methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)py-
ridine-2,4-dicarboxamide
Example 56:
(R*)-6-(Methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H-pyrazol-4-yl)py-
ridine-2,4-dicarboxamide
##STR00080##
[0412]
(+/-)-6-(Methoxy(phenyl)methyl)-N.sup.2-methyl-N.sup.4-(1H-pyrazol--
4-yl)pyridine-2,4-dicarboxamide (for a preparation see Example 54)
(90 mg) was purified by chiral HPLC. The racemate was dissolved in
EtOH (2 mL). Injection: 1.5 mL of the solution was injected onto
the column (20% EtOH (+0.2% isopropylamine)/heptane (+0.2%
isopropylamine), flow rate=30 mL/min, detection wavelength=215 nm,
4. Ref 550, 100, Column 30 mm.times.25 cm Chiralcel OD-H (5 .mu.m),
lot no. ODH11158-01). Total number of injections=1. Fractions from
9-11 min were bulked and labelled peak 1. Fractions from 12.5-17
min were bulked and labelled peak 2. The bulked pure fractions were
concentrated in vacuo and then transferred to weighed flasks.
[0413] The fractions corresponding to peak 1 were collected to
afford example 55 (35.4 mg)
[0414] LCMS (2 min Formic): Rt=0.83 min, [MH]+=366.2.
[0415] 1H NMR (400 MHz, DMSO-d6) .delta. ppm 12.69 (br. s., 1 H),
10.93 (s, 1 H) 8.67 (q, J=4.3 Hz, 1 H) 8.45 (d, J=1.7 Hz, 1 H),
8.17 (d, J=1.7 Hz, 1 H), 8.04 (br. s., 1 H), 7.72 (br. s., 1 H),
7.51 (d, J=7.1 Hz, 2 H), 7.33-7.39 (m, 2 H), 7.24-7.30 (m, 1 H),
5.52 (s, 1 H), 3.39 (s, 3 H), 2.88 (d, J=4.9 Hz, 3 H)
[0416] The fractions corresponding to peak 2 were collected to
afford example 56 (39.2 mg)
[0417] LCMS (2 min Formic): Rt=0.83 min, [MH]+=366.3.
Examples 57-76 and 81:
[0418] Examples 57-76 and 81 were prepared in an analogous manner
to other examples described above.
TABLE-US-00008 Rt Ex No. Name Structure [MH].sup.+ (min) 57
6-Benzyl-N.sup.4-ethyl-N.sup.2- methylpyridine-2,4- dicarboxamide
##STR00081## 298.3 0.91 (formic) 58 6-Benzyl-N.sup.4-(2-
methoxyethyl)-N.sup.2- methylpyridine-2,4- dicarboxamide
##STR00082## 328.2 0.91 (High pH) 59
6-(3-Methoxybenzyl)-N.sup.4-(2- methoxyethyl)-N.sup.2-
methylpyridine-2,4- dicarboxamide ##STR00083## 358.3 0.91 (High pH)
60 6-Benzyl-N.sup.4-isopropyl-N.sup.2- methylpyridine-2,4-
dicarboxamide ##STR00084## 312.2 0.97 (High pH) 61
(+/-)-6-Benzyl-N.sup.4-(2- hydroxypropyl)-N.sup.2-
methylpyridine-2,4- dicarboxamide ##STR00085## 328.1 0.80 (formic)
62 6-Benzyl-N.sup.4-(2-hydroxyethyl)- N.sup.2-methylpyridine-2,4-
dicarboxamide ##STR00086## 314.1 0.76 (formic) 63
6-Benzyl-N.sup.4-(3- (dimethylamino)propyl)-N.sup.2-
methylpyridine-2,4- dicarboxamide ##STR00087## 355.3 0.59 (formic)
64 N.sup.4-(3-Aminopropyl)-6-benzyl- N.sup.2-methylpyridine-2,4-
dicarboxamide hydrochloride ##STR00088## 327.3 0.55 (formic) 65
6-Benzyl-N.sup.4-isobutyl-N.sup.2- methylpyridine-2,4-
dicarboxamide ##STR00089## 326.3 1.07 (High pH) 66
6-Benzyl-N.sup.4-(tert-butyl)-N.sup.2- methylpyridine-2,4-
dicarboxamide ##STR00090## 326.3 1.10 (High pH) 67
6-Benzyl-N.sup.2-methyl-N.sup.4-(1- methyl-1H-pyrazol-5-
yl)pyridine-2,4-dicarboxamide ##STR00091## 350.3 0.90 (High pH) 68
(S)-N.sup.4-(2-(1H-1,2,4-Triazol-1- yl)ethyl)-N.sup.2-methyl-6-(1-
phenylethyl)pyridine-2,4- dicarboxamide ##STR00092## 379.4 0.87
(High pH) 69 (S)-N.sup.4-(2-(1H-Imidazol-1-
yl)ethyl)-N.sup.2-methyl-6-(1- phenylethyl)pyridine-2,4-
dicarboxamide ##STR00093## 378.4 0.90 (High pH) 70
(S)-N.sup.2-Methyl-6-(1- phenylethyl)-N.sup.4-(2-(thiazol-2-
yl)ethyl)pyridine-2,4- dicarboxamide ##STR00094## 395.4 1.01 (High
pH) 71 (S)-N.sup.2-Methyl-N.sup.4-(2-(2- methyl-1H-imidazol-1-
yl)ethyl)-6-(1- phenylethyl)pyridine-2,4- dicarboxamide
##STR00095## 392.4 0.91 (High pH) 72
(S)-N.sup.2-Methyl-N.sup.4-(4-methyl- 1H-pyrazol-3-yl)-6-(1-
phenylethyl)pyridine-2,4- dicarboxamide ##STR00096## 364.3 1.03
(formic) 73 (S)-N.sup.2-Methyl-N.sup.4-(3-methyl-
1H-pyrazol-4-yl)-6-(1- phenylethyl)pyridine-2,4- dicarboxamide
##STR00097## 364.4 0.93 (High pH) 74
(S)-N.sup.2-Methyl-N.sup.4-(3-methyl- 1,2,4-oxadiazol-5-yl)-6-(1-
phenylethyl)pyridine-2,4- dicarboxamide ##STR00098## 366.3 0.74
(High pH) 75 (S)-N.sup.2-Methyl-N.sup.4-(5-methyl-
1,3,4-oxadiazol-2-yl)-6-(1- phenylethyl)pyridine-2,4- dicarboxamide
##STR00099## 366.3 0.74 (High pH) 76
(S)-N.sup.2-Methyl-N.sup.4-(4-methyl- 4H-1,2,4-triazol-3-yl)-6-(1-
phenylethyl)pyridine-2,4- dicarboxamide ##STR00100## 365.3 0.77
(High pH) 81 6-Benzyl-N.sup.2-methylpyridine- 2,4-dicarboxamide
##STR00101## 270.1 0.78 (Formic)
Biological Data
[0419] The compounds of formula (I) may be tested in one or more of
the following assays:
Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
Assay
[0420] Bromodomain binding was assessed utilising a time resolved
fluorescent resonance energy transfer (TR-FRET) competition assay.
To enable this approach a known, high affinity, pan-BET interacting
small molecule was labelled with Alexa Fluor.RTM. 647, which is a
far-red-fluorescent dye (Reference Compound X). Reference Compound
X acts as a reporter of bromodomain binding and is the acceptor
fluorophore component of the TR-FRET pair. Europium chelate,
conjugated to an anti-6*His antibody, was utilised as the donor
fluorophore in the TR-FRET pair. The anti-6*His antibody binds
selectively to a six Histidine purification epitope added to the
amino-terminus of each of the BET tandem bromodomain protein
constructs used in this study. A TR-FRET signal is generated when
the donor and acceptor fluorophores are in close proximity, between
20-80 .ANG., which is enabled in this assay by binding of Reference
Compound X to the bromodomain protein.
[0421] Reference Compound X:
4-((Z)-3-(6-((5-(2-((4S)-6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f-
][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl)acetamido)pentyl)amino)-6-oxohex-
yl)-2-((2E,4E)-5-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indol-1-ium-2-y-
l)penta-2,4-dien-1-ylidene)-3-methyl-5-sulfoindolin-1-yl)butane-1-sulphona-
te)
##STR00102##
[0422] To a solution of
N-(5-aminopentyl)-2-((4S)-6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl)acetamide (for a
preparation see Reference Compound J, WO2011/054848A1, 1.7 mg, 3.53
.mu.mol) in DMF (40 .mu.L) was added a solution of
AlexaFluor647-ONSu (2.16 mg, 1.966 .mu.mol) also in DMF (100
.mu.L). The mixture was basified with DIPEA (1 .mu.l, 5.73 .mu.mol)
and agitated overnight on a vortex mixer.
[0423] The reaction mixture was evaporated to dryness. The solid
was dissolved in MeCN/water/AcOH (5/4/1, <1 mL) filtered and was
applied to a Phenomenex Jupiter C18 preparative column and eluted
with the following gradient (A=0.1% trifluoroacetic acid in water,
B=0.1% TFA/90% MeCN/10% water): Flow rate=10 mL/min., AU=20/10
(214nm):
[0424] 5-35%, t=0 min: B=5%; t=10 min: B=5%; t=100 min: B=35%;
t=115 min: B=100% (Sep. grad: 0.33%/min)
[0425] The major component was eluted over the range 26-28% B but
appeared to be composed of two peaks. The middle fraction (F1.26)
which should contain "both" components was analysed by analytical
HPLC (Spherisorb ODS2, 1 to 35% over 60 min): single component
eluting at 28%B.
[0426] Fractions F1.25/26&27 were combined and evaporated to
dryness. Transfered with DMF, evaporated to dryness, triturated
with dry ether and the blue solid dried overnight at <0.2 mbar:
1.54 mg.
[0427] Analytical HPLC (Sphersisorb ODS2, 1 to 35% B over 60 min):
MSM10520-1: [M+H].sup.+ (obs): 661.8/.sup.- corresponding with
M-29. This equates to [(M+2H)/2].sup.+ for a calculated mass of
1320.984 which is M-29. This is a standard occurence with the Alexa
Fluor 647 dye and represents a theoretical loss of two methylene
groups under the conditions of the mass spectrometer.
[0428] Assay Principle: In order to generate a TR-FRET signal,
donor fluorophore is excited by a laser at .lamda.337 nm, which
subsequently leads to emission at .lamda.618 nm. If the acceptor
fluorophore is in close proximity then energy transfer can occur,
which leads to emission of Alexa Fluor.RTM. 647 at .lamda.665 nm.
In the presence of competitor compound, Reference Compound X can be
displaced from binding to the bromodomain. If displacement occurs,
the acceptor fluorophore is no longer in proximity to the donor
fluorophore, which prevents fluorescent energy transfer and,
subsequently, a loss of Alexa Fluor.RTM. 647 emission at .lamda.665
nm.
[0429] The competition of the compounds of formula (I) with
Reference Compound X for binding to the BET family (BRD2, BRD3,
BRD4 and BRDT) was assessed using protein truncates spanning both
bromodomain 1 (BD1) and bromodomain 2 (BD2). In order to monitor
differential binding to either BD1 or BD2, single residue mutations
of key tyrosines to alanine were made in the acetyl lysine binding
pockets. To validate this approach, a double residue mutant tandem
domain protein was produced for each of the BET family members.
Utilising a Fluorescence Polarisation approach, binding affinities
for each of the single and double mutants for Reference Compound X
were determined. The affinities of the double mutant tandem
proteins for Reference Compound X were greatly greatly reduced in
comparison to the non mutated, wild type tandem BET proteins
(>1000 fold reduction in Kd). The affinities of the single
mutated bromdomain tandem proteins for Reference Compound X were
equi-potent with the corresponding non-mutated BET protein. These
data demonstrated that single mutations of Tyrosine to Alanine
reduce the Kd of the interaction between the mutated bromodomain
and Reference Compound X by >1000 fold. In the TR-FRET
competition assay, Reference Compound X is used at a concentration
that is equivalent to the Kd for the non-mutated bromodomain, which
ensures that no binding at the mutated bromodomain is detected.
[0430] Protein production: Recombinant Human Bromodomains [(BRD2
(1-473) (Y113A) and (Y386A), BRD3 (1-435) (Y73A) and (Y348A) BRD4
(1-477) (Y97A) and (Y390A) and BRDT (1-397) (Y66A) and (Y309A)]
were expressed in E. coli cells (in pET15b vector for BRD2/3/4 and
in pET28a vector for BRDT) with a 6-His tag at the N-terminal. The
His-tagged Bromodomain pellet was resuspended in 50 mM HEPES (pH
7.5), 300 mM NaCl, 10 mM imidazole & 1 .mu.L/mL protease
inhibitor cocktail and extracted from the E. coli cells using
sonication and purified using a nickel sepharose high performance
column, the proteins were washed and then eluted with a linear
gradient of 0-500 mM imidazole with buffer 50 mM HEPES (pH 7.5),
150 mM NaCl, 500 mM imidazole, over 20 column volumes. Final
purification was completed by Superdex 200 prep grade size
exclusion column. Purified protein was stored at -80.degree. C. in
20 mM HEPES pH 7.5 and 100 mM NaCl. Protein identity was confirmed
by peptide mass fingerprinting and predicted molecular weight
confirmed by mass spectrometry.
[0431] Protocol for Bromodomain BRD2, 3, 4 and T, BD1+BD2 Mutant
TR-FRET Competition Assays:
[0432] All assay components were dissolved in an assay buffer
composing of 50 mM HEPES pH 7.4, 50 mM NaCl, 5% Glycerol, 1 mM DTT
and 1 mM CHAPS. Reference Compound X was diluted, in assay buffer
containing 20 nM single mutant, tandem bromodomain protein, to a
concentration equivalent to 2*Kd for this bromodomain. The solution
containing bromodomain and Reference Compound X was added to dose
response dilutions of test compound or DMSO vehicle (a maximum of
0.5% DMSO is used in this assay) in Greiner 384 well black low
volume microtitre plates and subsequently incubated for 30 minutes
at room temperature. An equal volume of 3 nM of anti-6*His Europium
chelate was added to all wells, followed by a further 30 minute
incubation at room temperature. TR-FRET was detected using a Perkin
Elmer Multimode plate reader, by exciting the donor fluorophore at
.lamda.337 nm and subsequently, after a delay of 50 .mu.secs,
measuring emission of the donor and acceptor fluorophores at
.lamda.615 nm and .lamda.665 nm, respectively. In order to control
these assays, 16 replicates each of uninhibited (DMSO vehicle) and
inhibited (10*IC.sub.50 concentrations of Example 11 of WO
2011/054846A1) TR-FRET assays were included on every microtitre
plate.
[0433] cA four parameter curve fit of the following form was then
applied:
y=a+((b-a)/(1+(10{circumflex over ( )}x/10{circumflex over (
)}c){circumflex over ( )}d)
[0434] Where `a` is the minimum, `b` is the Hill slope, `c` is the
pIC.sub.50 and `d is the maximum.
[0435] All compounds (Examples) were each tested in the BRD4 BD1
and the BRD4 BD2 TR-FRET assays essentially as described above.
Those of skill in the art will recognise that in vitro binding
assays and cell-based assays for functional activity are subject to
experimental variability. Accordingly, it is to be understood that
the pIC.sub.50 values given below are exemplary only. pIC.sub.50
values are expressed as log.sub.10 units.
[0436] All Examples, with the exemption of Example 72, were found
to have a pIC.sub.50.gtoreq.5.0 in at least one assay described
above.
[0437] Examples 59, 61, 66 and 74-76 were found to have a
pIC.sub.50.gtoreq.5.0 and <6.0 in the BRD4 BD2 assay.
[0438] All other tested compounds were found to have a
pIC.sub.50.gtoreq.6.0 and <8.1 in the BRD4 BD2 assay. In
particular, Example 11 was found to have a pIC.sub.50 of 7.8 (n=2)
in the BRD4 BD2 assay; Example 12 was found to have a pIC.sub.50 of
7.7 (n=8) in the BRD4 BD2 assay; Example 16 was found to have a
pIC.sub.50 of 8.0 (n=5) in the BRD4 BD2 assay; and Example 55 was
found to have a pIC.sub.50 of 7.7 (n=3) in the BRD4 BD2 assay.
[0439] Calculation of Selectivity for BRD4 BD2 Over BRD4 BD1
[0440] Selectivity for BRD4 BD2 over BRD4 BD1 was calculated as
follows:
Selectivity=BRD4 BD2 pIC.sub.50-BRD4 BD1 pIC.sub.50
[0441] All Examples, with the exemption of Examples 62 and 72, were
found to have selectivity for BRD4 BD2 over BRD4 BD1 of .gtoreq.1
log unit in at least one of the TR-FRET assays described above, and
hence are at least 10 fold selective for BRD4 BD2 over BRD4
BD1.
[0442] Examples 1 to 56 and 78-80 were found to have selectivity
for BRD4 BD2 over BRD4 BD1 of .gtoreq.2 log unit in at least one of
the TR-FRET assays described above, and hence are at least 100 fold
selective for BRD4 BD2 over BRD4 BD1.
[0443] Example 11, 12 and 55 were found to have selectivity for
BRD4 BD2 over BRD4 BD1 of 2.7 log units in at least one of the
TR-FRET assays described above.
[0444] Example 16 was found to have a selectivity for BRD4 BD2 over
BRD4 BD1 of 3.0 log units in at least one of the TR-FRET assays
described above, and hence 1000-fold selective for BRD4 BD2 over
BRD4 BD1.
* * * * *